Field of the Invention
[0001] The present invention relates to a novel protease 3D structure, as well as variants
of a parent protease, in particular variants of amended properties, such as improved
thermostability and/or amended temperature activity profile. The invention also relates
to DNA sequences encoding such variants, their production in a recombinant host cell,
as well as methods of using the variants, in particular within the field of animal
feed and detergents. The invention furthermore relates to methods of generating and
preparing protease variants of amended properties. Preferred parent proteases are
Nocardiopsis proteases, such as proteases comprising the mature peptide parts of SEQ
ID NOs: 2, 4, 6, 8, 10, and 21.
Background of the Invention
[0003] JP 2003284571-A discloses, as SEQ ID NOs: 2 and 1, the amino acid sequence and the corresponding
DNA sequence, respectively, of a protease derived from Nocardiopsis sp. TOA-1 (FERM
P-18676). The sequences have been entered in the GENESEQ database as GENESEQP no.
ADF43564, and GENESEQN no. ADF43563, respectively.
[0004] JP 2-255081-A discloses a protease derived from Nocardiopsis sp. strain OPC-210 (FERM P-10508),
however without sequence information. The strain is no longer available, as the deposit
was withdrawn.
[0005] DD 200432|8 discloses a proteolytic preparation derived from Nocardiopsis dassonvillei strain
ZIMET 43647, however without sequence information. The strain appears to be no longer
available.
[0006] Additional Nocardiopsis protease sequences are disclosed in
PCT/DK04/000433 ("Protease 08," SEQ ID NOs: 9-10 herein);
PCT/DK04/000434 ("Protease 11," SEQ ID NOs: 5-6 herein);
PCT/DK04/000432 ("Protease 18," S E Q ID NOs: 3-4 herein); and
PCT/DK04/000435 ("Protease 35," SEQ ID NOs: 7-8 herein).
[0007] It is an object of the present invention to provide alternative proteases, in particular
for use in animal feed and/or detergents, in particular novel and improved protease
variants, preferably of amended properties, such as improved thermostability and/or
a higher or lower optimum temperature.
Summary of the Invention
[0008] The present invention relates to a variant of a parent protease, comprising a substitution
in at least one position of at least one region selected from the group of regions
consisting of: 6-18; 22-28; 32-39; 42-58; 62-63; 66-76; 78-100; 103-106; 111-114;
118-131; 134-136; 139-141; 144-151; 155-156; 160-176; 179-181; and 184-188; wherein
- (a) the variant has protease activity; and
- (b) each position corresponds to a position of amino acids 1 to 188 of SEQ ID NO:
2; and
- (c) the variant has a percentage of identity to amino acids 1 to 188 of SEQ ID NO:
2 of at least 60%.
[0009] The present invention also relates to isolated nucleic acid sequences encoding the
protease variant and to nucleic acid constructs, vectors, and host cells comprising
the nucleic acid sequences as well as methods for producing and using the protease
variants.
Brief Description of the Figures
[0010]
Figure 1 is a multiple alignment of Protease 10, Protease 18, Protease 11, Protease
35 and Protease 08 (the mature peptide parts of SEQ ID NOs: 2, 4, 6, 8 and 10, respectively),
also including a protease variant of the invention, viz. Protease 22 (amino acids
1-188 of SEQ ID NO: 21); and
Figure 2 provides the coordinates of the novel 3D structure of Protease 10 (amino
acids 1 to 188 of SEQ ID NO: 2) derived from Nocardiopsis sp. NRRL 18262.
Detailed Description of the Invention
Three-dimensional Structure of Protease 10
[0011] The structure of Protease 10 was solved in accordance with the principles for X-ray
crystallographic methods as given, for example, in
X-Ray Structure Determination, Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc.
NY, 1989. The structural coordinates for the crystal structure at 2.2 A resolution using the
isomorphous replacement method are given in Fig. 2 in standard PDB format (Protein
Data Bank, Brookhaven National Laboratory, Brookhaven, CT). The PDB file of Fig. 2
relates to the mature peptide part of Protease 10 corresponding to residues 1-188
of SEQ ID NO: 2.
Molecular Dynamics (MD)
[0012] Molecular Dynamics (MD) simulations are indicative of the mobility of the amino acids
in a protein structure (see
McCammon, JA and Harvey, SC., (1987), "Dynamics of proteins and nucleic acids", Cambridge
University Press). Such protein dynamics are often compared to the crystallographic B-factors (see
Stout, GH and Jensen, LH, (1989), "X-ray structure determination", Wiley). By running the MD simulation at, e.g., different temperatures, the temperature
related mobility of residues is simulated. Regions having the highest mobility or
flexibility (here isotropic fluctuations) may be suggested for random mutagenesis.
It is here understood that the high mobility found in certain areas of the protein,
may be thermally improved by substituting these residues.
[0013] Using the programs CHARMM (Accelrys) and NAMD (University of Illinois at Urbana-Champaign)
the Protease 10 structure described above was subjected to MD at 300 and 400K. Starting
from the coordinates of Figure 2 hydrogen and missing heavy atoms were built using
CHARMM procedures HBUILD and IC BUILD respectively. Then the structure was minimized
using CHARMM Conjugate Gradients (CONJ) minimization procedure for a total of 200
steps. The protein was then put on a 70 X 70 X 70 Angstrom box and solvated with TIP3
water molecules. A total of 11124 water molecules were added and then minimized, keeping
the protein coordinates fixed, using CHARMM Adopted Basis Newton Raphson (ABNR) minimization
procedure for 20000 steps. The system was then heated to the desired temperature at
a rate of 1K every 100 steps using the NAMD software. After an equilibration of 50
picoseconds, an NVE ensemble MD was run for 1 nanosecond, both steps done with the
software NAMD. A cut-off of 12 Angstrom was used for the non-bonded interactions.
Periodic boundary conditions were used after the solvation step and for all the subsequent
ones. The isotropic root mean square (RMS) fluctuations were calculated with the CHARMM
procedure COOR DYNA.
[0014] The following suggested regions for mutagenesis result from MD simulations: From
residue 160 to 170, from residue 78 to 90, from residue 43 to 50, from residue 66
to 75, and from residue 22 to 28.
Strategy for Preparing Variants
[0015] Regions of amino acid residues, as well as individual amino acid substitutions, were
suggested for mutagenesis based on the 3D-structure of Fig. 2 and the alignment of
the five known proteases (upper five rows of Fig. 1), mainly with a view to improving
thermostability.
[0016] The following regions were suggested, cf. claim 1: 6-18; 22-28; 32-39; 42-58; 62-63;
66-76; 78-100; 103-106; 111-114; 118-131; 134-136; 139-141; 144-151; 155-156; 160-176;
179-181; and 184-188.
[0017] At least one of the following positions of the above regions are preferably subjected
to mutagenesis, cf. claim 3; 6; 7; 8; 9; 10; 12; 13; 16; 17; 18; 22; 23; 24; 25; 26;
27; 28; 32; 33; 37; 38; 39; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55;
56; 58; 62; 63; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 78; 79; 80; 81; 82; 83;
84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 103; 105; 106;
111; 113; 114; 118; 120; 122; 124; 125; 127; 129; 130; 131; 134; 135; 136; 139; 140;
141; 144; 145; 146; 147; 148; 149; 150; 151; 155; 156; 160; 161; 162; 163; 164; 165;
166; 167; 168; 169; 170; 171; 172; 173; 174; 175; 176; 179; 180; 181; 184; 185; 186;
187; and/or 188.
[0018] Contemplated specific variants are listed in the claims, viz. variants of Protease
10, Protease 18, Protease 11, Protease 35 as well as Protease 08 in claims 4 and 15;
variants of Protease 10 in claim 16; variants of Protease 18 in claim 17; variants
of Protease 11 in claim 18; variants of Protease 35 in claim 19; and variants of Protease
08 in claim 20.
[0019] The various concepts underlying the invention are also reflected in the claims as
follows: Stabilization by disulfide-bridges in claims 5 and 6; proline-stabilization
in claims 7-8; substitution of exposed neutral residues with negatively charged residues
in claims 9-10; substitution of exposed neutral residues with positively charged residues
in claims 11-12; substitution of small residues with bulkier residues inside the protein
in claim 13; and regions proposed for mutagenesis following MD simulations in claim
14.
[0020] The term "at least one" means "one or more," viz., e.g. in the context of regions:
One, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen, or seventeen; or, in the context of positions or substitutions:
One, two, three, four, five, and so on, up to e.g. ninety.
[0021] In a particular embodiment, the number of regions proposed for and/or subjected to
mutagenesis is at least one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, or at least seventeen.
[0022] In another particular embodiment, the number of regions proposed for and/or subjected
to mutagenesis is no more than one, two, three, four, five, six, seven, eight, nine,
ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or no more than seventeen.
Polypeptides Having Protease Activity
[0023] Polypeptides having protease activity, or proteases, are sometimes also designated
peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Proteases may
be of the exo-type that hydrolyse peptides starting at either end thereof, or of the
endo-type that act internally in polypeptide chains (endopeptidases). Endopeptidases
show activity on N- and C-terminally blocked peptide substrates that are relevant
for the specificity of the protease in question.
[0024] The term "protease" is defined herein as an enzyme that hydrolyses peptide bonds.
This definition of protease also applies to the protease-part of the terms "parent
protease" and "protease variant," as used herein. The term "protease" includes any
enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses
thereof). The EC number refers to
Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in
Eur. J. Bio-chem. 1994, 223, 1-5;
Eur. J. Biochem. 1995, 232, 1-6;
Eur. J. Biochem. 1996, 237, 1-5;
Eur. J. Biochem. 1997, 250, 1-6; and
Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. the
World Wide Web (WWW) at http://www.chem.qmw.ac.uk/iubmb/enzyme/index.html.
[0025] Proteases are classified on the basis of their catalytic mechanism into the following
groups: Serine proteases (S), Cysteine proteases (C), Aspartic proteases (A), Metallo
proteases (M), and Unknown, or as yet unclassified, proteases (U), see
Handbook of Proteolytic Enzymes, A.J.Barrett, N.D.Rawlings, J.F.Woessner (eds), Academic
Press (1998), in particular the general introduction part.
[0026] In particular embodiments, the parent proteases and/or the protease variants of the
invention and for use according to the invention are selected from the group consisting
of:
(a) Proteases belonging to the EC 3.4.-.- enzyme group;
(b) Serine proteases belonging to the S group of the above Handbook;
(c1) Serine proteases of peptidase family S2A; and
(c2) Serine proteases of peptidase family S1 E as described in Biochem.J. 290:205-218 (1993) and in MEROPS protease database, release 6.20, March 24, 2003, (www.merops.ac.uk).
The database is described in Rawlings, N.D., O'Brien, E. A. & Barrett, A.J. (2002) MEROPS: the protease database.
Nucleic Acids Res. 30, 343-346.
[0027] For determining whether a given protease is a Serine protease, and a family S2A protease,
reference is made to the above Handbook and the principles indicated therein. Such
determination can be carried out for all types of proteases, be it naturally occurring
or wild-type proteases; or genetically engineered or synthetic proteases.
[0028] Protease activity can be measured using any assay, in which a substrate is employed,
that includes peptide bonds relevant for the specificity of the protease in question.
Assay-pH and assay-temperature are likewise to be adapted to the protease in question.
Examples of assay-pH-values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples
of assay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C.
Examples of protease substrates are casein, such as Azurine-Crosslinked Casein (AZCL-casein).
Examples of suitable protease assays are described in the experimental part.
Parent Protease
[0029] The parent protease is a protease from which the protease variant is, or can be,
derived. For the present purposes, any protease can be used as the parent protease,
as long as the resulting protease variant is homologous to Protease 10, i.e. the protease
derived from Nocardiopsis sp. NRRL 18262 and comprising amino acids 1-188 of SEQ ID
NO: 2.
[0030] In a particular embodiment the parent protease is also homologous to Protease 10.
[0031] In the present context, homologous means having an identity of at least 60% to SEQ
ID NO: 2, viz. amino acids 1-188 of the mature peptide part of Protease 10. Homology
is determined as generally described below in the section entitled Amino Acid Homology.
[0032] The parent protease may be a wild-type or naturally occurring polypeptide, or an
allelic variant thereof, or a fragment thereof that has protease acticity, in particular
a mature part thereof. It may also be a variant thereof and/or a genetically engineered
or synthetic polypeptide.
[0033] In a particular embodiment the wild-type parent protease is i) a bacterial protease;
ii) a protease of the phylum Actinobacteria; iii) of the class Actinobacteria; iv)
of the order Actinomycetales v) of the family Nocardiopsaceae; vi) of the genus Nocardiopsis;
and/or a protease derived from vii) Nocardiopsis species, such as Nocardiopsis alba,
Nocardiopsis antarctica, Nocardiopsis composta, Nocardiopsis dassonvillei, Nocardiopsis
exhalans, Nocardiopsis halophila, Nocardiopsis halotolerans, Nocardiopsis kunsanensis,
Nocardiopsis listeri, Nocardiopsis lucentensis, Nocardiopsis metallicus, Nocardiopsis
prasina, Nocardiopsis sp., Nocardiopsis synnemataformans, Nocardiopsis trehalosi,
Nocardiopsis tropica, Nocardiopsis umidischolae, or Nocardiopsis xinjiangensis.
[0034] Examples of such strains are: Nocardiopsis alba DSM 15647 (wild-type producer of
Protease 08), Nocardiopsis dassonvillei NRRL 18133 (wild-type producer of Protease
M58-1 described in
WO 88/03947), Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (wild-type producer of
Protease 18), Nocardiopsis prasina DSM 15648 (wild-type producer of Protease 11),
Nocardiopsis prasina DSM 15649 (wild-type producer of Protease 35), Nocardiopsis sp.
NRRL 18262 (wild-type producer of Protease 10), Nocardiopsis sp. FERM P-18676 (described
in
JP 2003284571-A).
[0035] Strains of these species are accessible to the public in a number of culture collections,
such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural
Research Service Patent Culture Collection, Northern Regional Research Center (NRRL),
e.g. Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 is publicly available
from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,
Germany).
[0036] Furthermore, such polypeptides may be identified and obtained from other sources
including microorganisms or DNA isolated from nature (e.g., soil, composts, water,
etc.) using suitable probes. Techniques for isolating microorganisms or DNA from natural
habitats are well known in the art. The nucleic acid sequence may then be derived
by similarly screening a genomic or cDNA library of another microorganism. Once a
nucleic acid sequence encoding a polypeptide has been detected with the probe(s),
the sequence may be isolated or cloned by utilizing techniques which are known to
those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
[0037] The parent protease may be a mature part of any of the amino acid sequences referred
to above. A mature part means a mature amino acid sequence and refers to that part
of an amino acid sequence which remains after a potential signal peptide part and/or
propeptide part has been cleaved off. The mature parts of each of the proteases Protease
08, 10, 11, 18, 22 and 35 are specified in the appended sequence listing.
[0038] The parent protease may also be a fragment of a specified amino acid sequence, viz.
a polypeptide having one or more amino acids deleted from the amino and/or carboxyl
terminus of this amino acid sequence. In one embodiment, a fragment contains at least
80, or at least 90, or at least 100, or at least 110, or at least 120, or at least
130, or at least 140, or at least 150, or at least 160, or at least 170, or at least
180, or at least 185 amino acid residues.
[0039] The parent protease may also be an allelic variant, allelic referring to the existence
of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic
variation arises naturally through mutation, and may result in polymorphism within
populations. Gene mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An allelic variant of
a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0040] In another embodiment, the parent protease may be a genetically engineered protease,
e.g. a variant of the wild-type or natural parent proteases referred to above comprising
a substitution, deletion, and/or insertion of one or more amino acids. In other words:
The parent protease may itself be a protease variant, such as Protease 22. The amino
acid sequence of such parent protease may differ from the amino acid sequence specified
by an insertion or deletion of one or more amino acid residues and/or the substitution
of one or more amino acid residues by different amino acid residues. The amino acid
changes may be of a minor, or of a major, nature. Amino acid changes of a major nature
are e.g. those resulting in a variant protease of the present invention with amended
properties. In another particular embodiment, the amino acid changes are of a minor
nature, that is conservative amino acid substitutions that do not significantly affect
the folding and/or activity of the protein; small deletions, typically of one to about
30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue; a small linker peptide of up to about 20-25 residues; or a small
extension that facilitates purification by changing net charge or another function,
such as a poly-histidine tract, an antigenic epitope or a binding domain.
[0041] Examples of conservative substitutions are within the group of basic amino acids
(arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid),
polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine
and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small
amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions
which do not generally alter the specific activity are known in the art and are described,
for example, by
H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,
Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,
Ala/Glu, and Asp/Gly as well as these in reverse.
[0042] Still further examples of genetically engineered parent proteases are synthetic proteases,
designed by man, and expectedly not occurring in nature.
EP 897985 discloses a process of preparing a consensus protein. Shuffled proteases are other
examples of synthetic or genetically engineered parent proteases, which can be prepared
as is generally known in the art, eg by Site-directed Mutagenesis, by PCR (using a
PCR fragment containing the desired mutation as one of the primers in the PCR reactions),
or by Random Mutagenesis. Included in the concept of a synthetic protease is also
any hybrid or chimeric protease, i.e. a protease which comprises a combination of
partial amino acid sequences derived from at least two proteases. Gene shuffling is
generally described in e.g.
WO 95/22625 and
WO 96/00343. Re-combination of protease genes can be made independently of the specific sequence
of the parents by synthetic shuffling as described in
Ness, J.E. et al, in Nature Biotechnology, Vol. 20 (12), pp. 1251-1255, 2002. Synthetic oligonucleotides degenerated in their DNA sequence to provide the possibility
of all amino acids found in the set of parent proteases are designed and the genes
assembled according to the reference. The shuffling can be carried out for the full
length sequence or for only part of the sequence and then later combined with the
rest of the gene to give a full length sequence. Two, three, four, five or all six
of the the proteases designated Protease 10, 18, 11, 35, 08 and 22 (SEQ ID NOs: 2,
4, 6, 8, 10, and 21; in particular the mature parts thereof) are particular examples
of such parent proteases which can be subjected to shuffling as described above, to
provide additional proteases of the invention.
[0043] In further particular embodiments, the parent protease comprises, or consists of,
respectively, the amino acid sequence specified, or an allelic variant thereof; or
a fragment thereof that has protease activity.
[0044] In still further particular embodiments, the protease variant of the invention is
not identical to: (i) amino acids 1-188 of SEQ ID NO: 2, amino acids 1-188 of SEQ
ID NO: 4, amino acids 1-188 of SEQ ID NO: 6, amino acids 1-188 of SEQ ID NO: 8, and
amino acids 1-188 of SEQ ID NO: 10; (ii) amino acids 1-188 of SEQ ID NO: 2; (iii)
amino acids 1-188 of SEQ ID NO: 2 with the substitution T87A; (iv) amino acids 1-188
of SEQ ID NO: 4; (v) amino acids 1-188 of SEQ ID NO: 6; (vi) amino acids 1-188 of
SEQ ID NO: 8; (vii) amino acids 1-188 of SEQ ID NO: 10; (viii) the protease derived
from Nocardiopsis dassonvillei NRRL 18133; (ix) the protease having amino acids 1
to 188 of SEQ ID NO: 2 as disclosed in
JP 2003284571-A; (x) the protease having the sequence entered in GENESEQP with no. ADF43564; (xi)
the protease disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 2, in particular the mature part thereof; (xii) the protease disclosed
in
DK patent application no. 2004 00969 as SEQ ID NO: 4, in particular the mature part thereof; (xiii) the protease disclosed
in
DK patent application no. 2004 00969 as SEQ ID NO: 6, in particular the mature part thereof; (xiv) the protease disclosed
in
DK patent application no. 2004 00969 as SEQ ID NO: 8, in particular the mature part thereof; (xv) the protease disclosed
in
DK patent application no. 2004 00969 as SEQ ID NO: 10, in particular the mature part thereof; (xvi) the protease disclosed
in
DK patent application no. 2004 00969 as SEQ ID NO: 12, in particular the mature part thereof; and/or (xvii) any prior
art protease of a percentage of identity to SEQ ID NO: 2 of at least 60%.
Microorganism Taxonomy
Amino Acid Homology
[0046] The present invention refers to proteases, viz. parent proteases, and/or protease
variants, having a certain degree of identity to amino acids 1 to 188 of SEQ ID NO:
2, such parent and/or variant proteases being hereinafter designated "homologous proteases".
[0047] For purposes of the present invention the degree of identity between two amino acid
sequences, as well as the degree of identity between two nucleotide sequences, is
determined by the program "align" which is a Needleman-Wunsch alignment (i.e. a global
alignment). The program is used for alignment of polypeptide, as well as nucleotide
sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignments,
and the default identity matrix is used for nucleotide alignments. The penalty for
the first residue of a gap is -12 for polypeptides and -16 for nucleotides. The penalties
for further residues of a gap are -2 for polypeptides, and -4 for nucleotides.
[0050] In particular embodiments, the homologous protease has an amino acid sequence which
has a degree of identity to amino acids 1 to 188 of SEQ ID NO: 2 of at least 60%,
62%, 64%, 66%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
of at least about 99%.
[0051] In alternative embodiments, the homologous protease has an amino acid sequence which
has a degree of identity to SEQ ID NO: 2 of at least 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, or at least 59%.
[0052] In another particular embodiment, the parent protease, and/or the protease variant,
comprises a mature amino acid sequence which differs by no more than seventyfive,
seventyfour, seventythree, seventytwo, seventyone, seventy, sixtynine, sixtyeight,
sixtyseven, sixtysix, sixtyfive, sixtyfour, sixtythree, sixtytwo, sixtyone, sixty,
fiftynine, fiftyeight, fiftyseven, fiftysix, fiftyfive, fiftyfour, fiftythree, fiftytwo,
fiftyone, fifty, fortynine, fortyeight, fortyseven, fortysix, fortyfive, fortyfour,
fortythree, fortytwo, fortyone, forty, thirtynine, thirtyeight, thirtyseven, thirtysix,
thirtyfive, thirtyfour, thirtythree, thirtytwo, thirtyone, thirty, twentynine, twentyeight,
twentyseven, twentysix, twentyfive, twentyfour, twentythree, twentytwo, twentyone,
twenty, nineteen, eighteen, seventeen, sixteen, fifteen, fourteen, thirteen, twelve,
eleven, ten, nine, eight, seven, six, five, four, three, by no more than two, or only
by one amino acid(s) from the specified amino acid sequence, e.g. amino acids 1 to
188 of SEQ ID NO: 2.
[0053] In a still further particular embodiment, the parent protease, and/or the protease
variant, comprises a mature amino acid sequence which differs by at least seventyfive,
seventyfour, seventythree, seventytwo, seventyone, seventy, sixtynine, sixtyeight,
sixtyseven, sixtysix, sixtyfive, sixtyfour, sixtythree, sixtytwo, sixtyone, sixty,
fiftynine, fiftyeight, fiftyseven, fiftysix, fiftyfive, fiftyfour, fiftythree, fiftytwo,
fiftyone, fifty, fortynine, fortyeight, fortyseven, fortysix, fortyfive, fortyfour,
fortythree, fortytwo, fortyone, forty, thirtynine, thirtyeight, thirtyseven, thirtysix,
thirtyfive, thirtyfour, thirtythree, thirtytwo, thirtyone, thirty, twentynine, twentyeight,
twentyseven, twentysix, twentyfive, twentyfour, twentythree, twentytwo, twentyone,
twenty, nineteen, eighteen, seventeen, sixteen, fifteen, fourteen, thirteen, twelve,
eleven, ten, nine, eight, seven, six, five, four, three, by at least two, or by one
amino acid(s) from the specified amino acid sequence, e.g. amino acids 1 to 188 of
SEQ ID NO: 2.
Nucleic Acid Hybridization
[0054] In the alternative, homologous parent proteases, as well as variant proteases, may
be defined as being encoded by a nucleic acid sequence which hybridizes under very
low stringency conditions, preferably low stringency conditions, more preferably medium
stringency conditions, more preferably medium-high stringency conditions, even more
preferably high stringency conditions, and most preferably very high stringency conditions
with nucleotides 900-1466, or 900-1463, of SEQ ID NO: 1, or a subsequence or a complementary
strand thereof (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning,
A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). A subsequence may
be at least 100 nucleotides, or at least 200, 300, 400, or at least 500 nucleotides.
Moreover, the subsequence may encode a polypeptide fragment that has the relevant
enzyme activity.
[0055] For long probes of at least 100 nucleotides in length, very low to very high stringency
conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3%
SDS, 200 pg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for
very low and low stringencies, 35% formamide for medium and medium-high stringencies,
or 50% formamide for high and very high stringencies, following standard Southern
blotting procedures.
[0056] For long probes of at least 100 nucleotides in length, the carrier material is finally
washed three times each for 15 minutes using 2 x SSC, 0.2% SDS preferably at least
at 45°C (very low stringency), more preferably at least at 50°C (low stringency),
more preferably at least at 55°C (medium stringency), more preferably at least at
60°C (medium-high stringency), even more preferably at least at 65°C (high stringency),
and most preferably at least at 70°C (very high stringency).
[0057] For short probes which are about 15 nucleotides to about 70 nucleotides in length,
stringency conditions are defined as prehybridization, hybridization, and washing
post-hybridization at 5°C to 10°C below the calculated T
m using the calculation according to
Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt's solution,
1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg
of yeast RNA per ml following standard Southern blotting procedures.
[0058] For short probes which are about 15 nucleotides to about 70 nucleotides in length,
the carrier material is washed once in 6X SCC plus 0.1% SDS for 15 minutes and twice
each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T
m.
Position numbering
[0059] In the present context, the basis for numbering positions is amino acids 1 to 188
of SEQ ID NO: 2, Protease 10, starting with A1 and ending with T188, see Fig. 1. A
parent protease, as well as a variant protease, may comprise extensions as compared
to SEQ ID NO: 2, i.e. in the N-terminal, and/or the C-terminal ends thereof. The amino
acids of such extensions, if any, are to be numbered as is usual in the art, i.e.
for a C-terminal extension: 189, 190, 191 and so forth, and for an N-terminal extension
-1, -2, -3 and so forth.
Alterations, such as Substitutions, Deletions, Insertions
[0060] In the present context, the following are examples of various ways in which a protease
variant can be designed or derived from a parent amino acid sequence: An amino acid
can be substituted with another amino acid; an amino acid can be deleted; an amino
acid can be inserted; as well as any combination of any number of such alterations.
[0061] For the present purposes, the term substitution is intended to include any number
of any type of such alterations. This is a reasonable definition, because, for example,
a deletion can be regarded as a substitution of an amino acid, AA, in a given position,
nn, with nothing, (). Such substitution can be designated: AAnn(). Likewise, an insertion
of only one amino acid, BB, downstream an amino acid, AA, in a given position, nn,
can be designated: ()nnaBB. And if two amino acids, BB and CC, are inserted downstream
of amino acid AA in position nn, this substitution (combination of two substitutions)
can be designated: ()nnaBB+()nnbCC, the thus created gaps between amino acids nn and
nn+1 in the parent sequence being assigned lower case or subscript letters a, b, c
etc. to the former position number, here nn. A similar numbering procedure is followed
when aligning a new sequence to the multiple alignment of Fig. 1, in case of a gap
being created by the alignment between amino acids nn and nn+1: Each position of the
gap is assigned a number: nna, nnb etc.. A comma (,) between substituents, as e.g.
in the substitution T129E,D,Y,Q means "either or", i.e. that T129 is substituted with
E, or D, or Y, or Q. A plus-sign (+) between substitutions, e.g. 129D+135P means "and",
i.e. that these two single substitutions are combined in one and the same protease
variant.
[0062] In the present context, the term "a" substitution" means at least one substitution.
At least one means one or more, e.g. one, or two, or three, or four, or five, or six,
or seven, or eight, or nine, or ten, or twelve, or fourteen, or fifteen, or sixteen,
or eighteen, or twenty, or twentytwo or twentyfour, or twentyfive, or twenty eight,
or thirty, and so on, to include in principle, any number of substitutions. The variants
of the invention, however, still have to be, e.g., at least 60% identical to SEQ ID
NO: 2, this percentage being determined by the above-mentioned program. The substitutions
can be applied to any position encompassed by any region mentioned in claim 1, and
variants comprising combinations of any number and type of such substitutions are
also included. The term substitution as used herein also include deletions, as well
as extensions, or insertions, that may add to the length of the sequence corresponding
to amino acids 1 to 188 of SEQ ID NO: 2.
[0063] Furthermore, the term "a substitution" embraces a substitution into any one of the
other nineteen natural amino acids, or into other amino acids, such as non-natural
amino acids. For example, a substitution of amino acid T in position 22 includes each
of the following substitutions: 22A, 22C, 22D, 22E, 22F, 22G, 22H, 22I, 22K, 22L,
22M, 22N, 22P, 22Q, 22R, 22S, 22V, 22W, and 22Y. This is, by the way, equivalent to
the designation 22X, wherein X designates any amino acid. These substitutions can
also be designated T22A, T22C, T22X, etc. The same applies by analogy to each and
every position mentioned herein, to specifically include herein any one of such substitutions.
Identifying Corresponding Position Numbers
[0064] For each amino acid residue in each parent, or variant, protease of the invention,
and/or for use according to the invention, it is possible to directly and unambiguously
assign an amino acid residue in the sequence of amino acids 1 to 188 of SEQ ID NO:
2 to which it corresponds. Corresponding residues are assigned the same number, by
reference to the Protease 10 sequence.
[0065] As it appears from the numbering of Fig. 1, in conjunction with the numbering of
the sequence listing, for each amino acid residue of each of the proteases Protease
10, Protease 18, Protease 11, Protease 35, Protease 08, and Protease 22, the corresponding
amino acid residue in SEQ ID NO: 2 has the same number. This number is easily derivable
from Fig. 1. At least in case of these six proteases, the number is the same as the
number assigned to this amino acid residue in the sequence listing for the mature
part of the respective protease.
[0066] For a given position in another protease - be it a parent or a variant protease -
a corresponding position of SEQ ID NO: 2 can always be found, as follows:
The amino acid sequence of another parent protease, or, in turn, of a variant protease
amino acid sequence, is designated SEQ-X. A position corresponding to position N of
SEQ ID NO: 2 is found as follows: The parent or variant protease amino acid sequence
SEQ-X is aligned with SEQ ID NO: 2 as specified above in the section entitled Amino
Acid Homology. From the alignment, the position in sequence SEQ-X corresponding to
position N of SEQ ID NO: 2 can be clearly and unambiguously derived, using the principles
described below.
[0067] SEQ-X is the mature part of the protease in question. In the alternative, it may
also include a signal peptide part, and/or a propeptide part, or it may be a fragment
of the mature protease which has protease activity, e.g. a fragment of the same length
as SEQ ID NO: 2, and/or it may be the fragment which extends from A1 to T188 when
aligned with SEQ ID NO: 2 as described herein.
Region and Position
[0068] In the present context, the term region means at least one position of a parent protease
amino acid sequence, the term position designating an amino acid residue of such amino
acid sequence. In one embodiment, region means one or more successive positions of
the parent protease amino acid sequence, e.g. one, two, three, four, five, six, seven,
eight, etc., up to any number of consecutive positions of the sequence. Accordingly,
a region may consist of one position only, or it may consist of any number of consecutive
positions, such as, e.g., position no. 62 and 63; or position no. 111, 112, 113 and
114. For the present purposes, these two regions are designated 62-63, and 111-114,
respectively. The boundaries of these regions or ranges are included in the region.
[0069] A region encompasses specifically each and every position it embraces. For example,
region 111-114 specifically encompasses each of the positions 111, 112, 113, and 114.
The same applies by analogy for the other regions mentioned herein.
Thermostability
[0070] For the present purposes, the term thermostable as applied in the context of a certain
polypeptide, refers to the melting temperature, Tm, of such polypeptide, as determined
using Differential Scanning Calorimetry (DSC) in 10mM sodium phosphate, 50 mM sodium
chloride, pH 7.0, using a constant scan rate of 1.5°C/min.
[0071] The following Tm's were determined under the above conditions: 76.5°C (Protease 10),
83.0°C (Protease 18), 78.3°C (Protease 08), 76.6°C (Protease 35), 73.7°C (Protease
11), and 83.5°C (Protease 22).
[0072] For a thermostable polypeptide, the Tm is at least 83.1°C. In particular embodiments,
the Tm is at least 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
or at least 100°C.
[0073] In the alternative, the term thermostable refers to a melting temperature of at least
73.8, or at least 76.7°C, or at least 78.4°C, preferably at least 74, 75, 76, 77,
78, 79, 80, 81, 82, or at least 83°C, still as determined using DSC at a pH of 7.0.
[0074] For the determination of Tm, a sample of the polypeptide with a purity of at least
90% (or 91, 92, 93, 94, 95, 96, 97, or 98%) as determined by SDS-PAGE may be used.
Still further, the enzyme sample may have a concentration of between 0.5 and 2.5 mg/ml
protein (or between 0.6 and 2.4, or between 0.7 and 2.2, or between 0.8 and 2.0 mg/ml
protein), as determined from absorbance at 280 nm and based on an extinction coefficient
calculated from the amino acid sequence of the enzyme in question.
[0075] The DSC takes place at the desired pH (e.g. pH 5.5, 7.0, 3.0, or 2.5) and with a
constant heating rate, e.g. of 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C/min.
[0076] In a particular embodiment, the protease variant of the invention is thermostable,
preferably more thermostable than the parent protease. In this context, preferred
parent proteases are Protease 18, or Protease 10.
[0077] In another particular embodiment, a culture supernatant of the protease variant of
the invention, appropriately diluted, exhibits a residual activity after incubation
for four hours at 65°C in a 0.2M Na
2HPO
4 buffer, titrated with 0.1M citric acid to i) pH 6.0, or ii) pH 4.0, of at least 20%,
relative to an un-incubated (frozen) control, the activity being measured using the
Protazyme AK assay at pH 8.5 and 37°C, as described in Example 2. In further particular
embodiments, the residual activity is at least 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, or at least 77%.
Temperature Activity Profile
[0078] In a particular embodiment, the protease variant of the invention exhibits an amended
temperature activity profile as compared to, e.g., Protease 10 (or Protease 18, Protease
11, Protease 35, or Protease 08). For example, the protease variant of the invention
may exhibit a relative activity at pH 9 and 80°C of at least 0.40, preferably at least
0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, or at least 0.95, the
term "relative" referring to the maximum activity measured for the protease in question.
For Protease 22, the activity is relative to the activity at 80°C which is set to
1.000 (100%), and for Protease 10, the activity at 70°C is set to 1.000 (100%), see
Example 3. As another example, the protease variant of the invention exhibits a relative
activity at pH 9 and 90°C of at least 0.10, preferably at least 0.15, 0.20, 0.25,
0.30, or of at least 0.35. In a particular embodiment, the protease activity is measured
using the Protazyme AK assay of Example 1.
Low-allergenic Variants
[0079] In a specific embodiment, the protease variants of the present invention are (also)
low-allergenic variants, designed to invoke a reduced immunological response when
exposed to animals, including man. The term immunological response is to be understood
as any reaction by the immune system of an animal exposed to the protease variant.
One type of immunological response is an allergic response leading to increased levels
of IgE in the exposed animal. Low-allergenic variants may be prepared using techniques
known in the art. For example the protease variant may be conjugated with polymer
moieties shielding portions or epitopes of the protease variant involved in an immunological
response. Conjugation with polymers may involve in vitro chemical coupling of polymer
to the protease variant, e.g. as described in
WO 96/17929,
WO 98/30682,
WO 98/35026, and/or
WO 99/00489. Conjugation may in addition or alternatively thereto involve in vivo coupling of
polymers to the protease variant. Such conjugation may be achieved by genetic engineering
of the nucleotide sequence encoding the protease variant, inserting consensus sequences
encoding additional glycosylation sites in the protease variant and expressing the
protease variant in a host capable of glycosylating the protease variant, see e.g.
WO 00/26354. Another way of providing low-allergenic variants is genetic engineering of the nucleotide
sequence encoding the protease variant so as to cause the protease variants to self-oligomerize,
effecting that protease variant monomers may shield the epitopes of other protease
variant monomers and thereby lowering the antigenicity of the oligomers. Such products
and their preparation is described e.g. in
WO 96/16177. Epitopes involved in an immunological response may be identified by various methods
such as the phage display method described in
WO 00/26230 and
WO 01/83559, or the random approach described in
EP 561907. Once an epitope has been identified, its amino acid sequence may be altered to produce
altered immunological properties of the protease variant by known gene manipulation
techniques such as site directed mutagenesis (see e.g.
WO 00/26230,
WO 00/26354 and/or
WO 00/22103) and/or conjugation of a polymer may be done in sufficient proximity to the epitope
for the polymer to shield the epitope.
Nucleic Acid Sequences and Constructs
[0080] The present invention also relates to nucleic acid sequences comprising a nucleic
acid sequence which encodes a protease variant of the invention.
[0081] The term "isolated nucleic acid sequence" refers to a nucleic acid sequence which
is essentially free of other nucleic acid sequences, e.g., at least about 20% pure,
preferably at least about 40% pure, more preferably at least about 60% pure, even
more preferably at least about 80% pure, and most preferably at least about 90% pure
as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence
can be obtained by standard cloning procedures used in genetic engineering to relocate
the nucleic acid sequence from its natural location to a different site where it will
be reproduced. The cloning procedures may involve excision and isolation of a desired
nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide,
insertion of the fragment into a vector molecule, and incorporation of the recombinant
vector into a host cell where multiple copies or clones of the nucleic acid sequence
will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0082] The nucleic acid sequences of the invention can be prepared by introducing at least
one mutation into the parent protease coding sequence or a subsequence thereof, wherein
the mutant nucleic acid sequence encodes a variant protease. The introduction of a
mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide
may be accomplished by site-directed mutagenesis using any of the methods known in
the art, e.g. by site-directed mutagenesis, by random mutagenesis, or by doped, spiked,
or localized random mutagenesis.
[0083] Random mutagenesis is suitably performed either as localized or region-specific random
mutagenesis in at least three parts of the gene translating to the amino acid sequence
shown in question, or within the whole gene. When the mutagenesis is performed by
the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the
three non-parent nucleotides during the synthesis of the oligonucleotide at the positions
which are to be changed. The doping or spiking may be performed so that codons for
unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated
into the DNA encoding the protease enzyme by any technique, using, e.g., PCR, LCR
or any DNA polymerase and ligase as deemed appropriate.
[0084] Preferably, the doping is carried out using "constant random doping", in which the
percentage of wild-type and mutation in each position is predefined. Furthermore,
the doping may be directed toward a preference for the introduction of certain nucleotides,
and thereby a preference for the introduction of one or more specific amino acid residues.
The doping may be made, e.g., so as to allow for the introduction of 90% wild type
and 10% mutations in each position. An additional consideration in the choice of a
doping scheme is based on genetic as well as protein-structural constraints.
[0085] The random mutagenesis may be advantageously localized to a part of the parent protease
in question. This may, e.g., be advantageous when certain regions of the enzyme have
been identified to be of particular importance for a given property of the enzyme.
[0086] Alternative methods for providing variants of the invention include gene shuffling
e.g. as described in
WO 95/22625 or in
WO 96/00343, and the consensus derivation process as described in
EP 897985 (see the section "Parent Protease" for more details).
[0087] In particular embodiments, the nucleic acid sequence of the invention is not identical
to: (i) Nucleotides 900-1466, or 900-1463, of SEQ ID NO: 1, nucleotides 499-1062 of
SEQ ID NO: 3, nucleotides 496-1059 of SEQ ID NO: 5, nucleotides 496-1059 of SEQ ID
NO: 7, and nucleotides 502-1065 of SEQ ID NO: 9; (ii) nucleotides 900-1466 of SEQ
ID NO: 1; (iii) nucleotides 900-1463 of SEQ ID NO: 1; (iv) nucleotides 900-1463 of
SEQ ID NO: 1 as disclosed in
DK 1996 00013; (v) nucleotides 499-1062 of SEQ ID NO: 3; (vi) nucleotides 496-1059 of SEQ ID NO:
5; (vii) nucleotides 496-1059 of SEQ ID NO: 7; (viii) nucleotides 502-1065 of SEQ
ID NO: 9; (xi) the nucleic acid sequence encoding the mature peptide part of the protease
derived from Nocardiopsis dassonvillei NRRL 18133; (x) the nucleic acid sequence having
SEQ ID NO: 1 as disclosed in
JP 2003284571-A; (xi) the nucleic acid sequence GENESEQN no. ADF43563; (xii) the nucleic acid sequence
disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 1, in particular the mature peptide encoding part thereof; (xiii) the
nucleic acid sequencep disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 3, in particular the mature peptide encoding part thereof; (xiv) the
nucleic acid sequence disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 5, in particular the mature peptide encoding part thereof; (xv) the
nucleic acid sequence disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 7, in particular the mature peptide encoding part thereof; (xvi) the
nucleic acid sequence disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 9, in particular the mature peptide encoding part thereof; (xvii) the
nucleic acid sequence disclosed in
DK patent application no. 2004 00969 as SEQ ID NO: 11, in particular the mature peptide encoding part thereof; and/or
(xviii) nucleic acid sequences encoding any prior art proteases of at least 60% identity
to amino acids 1 to 188 of SEQ ID NO: 2.
Nucleic Acid Constructs
[0088] A nucleic acid construct comprises a nucleic acid sequence of the present invention
operably linked to one or more control sequences which direct the expression of the
coding sequence in a suitable host cell under conditions compatible with the control
sequences. Expression will be understood to include any step involved in the production
of the polypeptide including, but not limited to, transcription, post-transcriptional
modification, translation, post-translational modification, and secretion.
Expression vector
[0089] A nucleic acid sequence encoding a protease variant of the invention can be expressed
using an expression vector which typically includes control sequences encoding a promoter,
operator, ribosome binding site, translation initiation signal, and, optionally, a
repressor gene or various activator genes.
[0090] The recombinant expression vector carrying the DNA sequence encoding a protease variant
of the invention may be any vector which may conveniently be subjected to recombinant
DNA procedures, and the choice of vector will often depend on the host cell into which
it is to be introduced. The vector may be one which, when introduced into a host cell,
is integrated into the host cell genome and replicated together with the chromosome(s)
into which it has been integrated.
[0091] The protease variant may also be co-expressed together with at least one other enzyme
of animal feed interest, such as an alpha-amylase, a phytase, a galactanase, a xylanase,
an endoglucanase, an endo-1,3(4)-beta-glucanase, an alpha-galactosidase, and/or a
protease. The enzymes may be co-expressed from different vectors, from one vector,
or using a mixture of both techniques. When using different vectors, the vectors may
have different selectable markers, and different origins of replication. When using
only one vector, the genes can be expressed from one or more promoters. If cloned
under the regulation of one promoter (di- or multi-cistronic), the order in which
the genes are cloned may affect the expression levels of the proteins. The protease
variant may also be expressed as a fusion protein, i.e. that the gene encoding the
protease variant has been fused in frame to the gene encoding another protein. This
protein may be another enzyme or a functional domain from another enzyme.
Host Cells
[0092] The present invention also relates to recombinant host cells, comprising a nucleic
acid sequence of the invention, which are advantageously used in the recombinant production
of the polypeptides. A vector comprising a nucleic acid sequence of the present invention
is introduced into a host cell so that the vector is maintained as a chromosomal integrant
or as a self-replicating extra-chromosomal vector. The term "host cell" encompasses
any progeny of a parent cell that is not identical to the parent cell due to mutations
that occur during replication. The choice of a host cell will to a large extent depend
upon the gene encoding the polypeptide and its source.
[0093] The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular
microorganism, e.g., a eukaryote cell, such as an animal, a mammalian, an insect,
a plant, or a fungal cell. Preferred animal cells are non-human animal cells.
[0094] In a preferred embodiment, the host cell is a fungal cell, or a yeast cell, such
as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia cell. The fungal host cell may be a filamentous fungal cell, such as a
cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola,
Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
Useful unicellular cells are bacterial cells such as gram positive bacteria including,
but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and Bacillus thuringiensis, or a Streptomyces cell, such as Streptomyces
lividans or Streptomyces murinus, or a Nocardiopsis cell, or cells of lactic acid
bacteria; or gram negative bacteria such as E. coli and Pseudomonas sp. Lactic acid
bacteria include, but are not limited to, species of the genera Lactococcus, Lactobacillus,
Leuconostoc, Streptococcus, Pediococcus, and Enterococcus.
Methods of Production
[0095] The present invention also relates to methods for producing a protease variant of
the present invention comprising (a) cultivating a host cell under conditions conducive
for production of the protease variant; and (b) recovering the protease variant.
[0096] In the production methods of the present invention, the cells are cultivated in a
nutrient medium suitable for production of the polypeptide using methods known in
the art. For example, the cell may be cultivated by shake flask cultivation, small-scale
or large-scale fermentation (including continuous, batch, fed-batch, or solid state
fermentations) in laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the polypeptide to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen
sources and inorganic salts, using procedures known in the art. Suitable media are
available from commercial suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection). If the protease is
secreted into the nutrient medium, it can be recovered directly from the medium. If
it is not secreted, it can be recovered from cell lysates.
[0097] The resulting protease may be recovered by methods known in the art. For example,
it can be recovered from the nutrient medium by conventional procedures including,
but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation,
or precipitation.
[0098] The proteases of the present invention may be purified by a variety of procedures
known in the art including, but not limited to, chromatography (e.g., ion exchange,
affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification,
J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
Plants
[0099] The present invention also relates to a transgenic plant, plant part, or plant cell
which has been transformed with a nucleic acid sequence encoding a polypeptide having
protease activity of the present invention so as to express and produce the polypeptide
in recoverable quantities. The polypeptide may be recovered from the plant or plant
part. Alternatively, the plant or plant part containing the recombinant polypeptide
may be used as such for improving the quality of a food or feed, e.g., improving nutritional
value, palatability, and rheological properties, or to destroy an antinutritive factor.
[0101] The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot)
or engineered variants thereof. Examples of monocot plants are grasses, such as meadow
grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such
as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, triticale
(stabilized hybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examples
of dicot plants are tobacco, legumes, such as sunflower (Helianthus), cotton (Gossypium),
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family
Brassicaceae), such as cauliflower, rape seed, and the closely related model organism
Arabidopsis thaliana. Low-phytate plants as described e.g. in
US patent no. 5,689,054 and
US patent no. 6,111,168 are examples of engineered plants.
[0102] Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet,
pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower,
rape seed, and the closely related model organism Arabidopsis thaliana. Low-phytate
plants as described e.g. in
US patent no. 5,689,054 and
US patent no. 6,111,168 are examples of engineered plants. Examples of plant parts are stem, callus, leaves,
root, fruits, seeds, and tubers, as well as the individual tissues comprising these
parts, e.g. epidermis, mesophyll, parenchyma, vascular tissues, meristems. Also specific
plant cell compartments, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes,
and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever
the tissue origin, is considered to be a plant part. Likewise, plant parts such as
specific tissues and cells isolated to facilitate the utilisation of the invention
are also considered plant parts, e.g. embryos, endosperms, aleurone and seed coats.
[0103] Also included within the scope of the present invention are the progeny of such plants,
plant parts and plant cells.
[0104] The transgenic plant or plant cell expressing a polypeptide of the present invention
may be constructed in accordance with methods known in the art. Briefly, the plant
or plant cell is constructed by incorporating one or more expression constructs encoding
a polypeptide of the present invention into the plant host genome and propagating
the resulting modified plant or plant cell into a transgenic plant or plant cell.
[0105] Conveniently, the expression construct is a nucleic acid construct which comprises
a nucleic acid sequence encoding a polypeptide of the present invention operably linked
with appropriate regulatory sequences required for expression of the nucleic acid
sequence in the plant or plant part of choice. Furthermore, the expression construct
may comprise a selectable marker useful for identifying host cells into which the
expression construct has been integrated and DNA sequences necessary for introduction
of the construct into the plant in question (the latter depends on the DNA introduction
method to be used).
[0106] The choice of regulatory sequences, such as promoter and terminator sequences and
optionally signal or transit sequences are determined, for example, on the basis of
when, where, and how the polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present invention may be constitutive
or inducible, or may be developmental, stage or tissue specific, and the gene product
may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory
sequences are, for example, described by
Tague et al., 1988, Plant Physiology 86: 506.
[0107] For constitutive expression, the following promoters may be used: The 35S-CaMV promoter
(
Franck et al., 1980, Cell 21: 285-294), the maize ubiquitin 1 (
Christensen AH, Sharrock RA and Quail 1992. Maize polyubiquitin genes: structure,
thermal perturbation of expression and transcript splicing, and promoter activity
following transfer to protoplasts by electroporation), or the rice actin 1 promoter
(Plant Mo. Biol. 18, 675-689.;
Zhang W, McElroy D. and Wu R 1991, Analysis of rice Act1 5' region activity in transgenic
rice plants. Plant Cell 3, 1155-1165). Organ-specific promoters may be, for example, a promoter from storage sink tissues
such as seeds, potato tubers, and fruits (
Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (
Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter
from rice (
Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (
Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (
Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific
promoter known in the art, e.g., as described in
WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter
from rice or tomato (
Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (
Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (
Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (
Xu et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter may be inducible by abiotic treatments such as temperature,
drought or alterations in salinity or inducible by exogenously applied substances
that activate the promoter, e.g. ethanol, oestrogens, plant hormones like ethylene,
abscisic acid, gibberellic acid, and/or heavy metals.
[0108] A promoter enhancer element may also be used to achieve higher expression of the
enzyme in the plant. For instance, the promoter enhancer element may be an intron
which is placed between the promoter and the nucleotide sequence encoding a polypeptide
of the present invention. For instance, Xu et al., 1993, supra disclose the use of
the first intron of the rice actin 1 gene to enhance expression.
[0109] Still further, the codon usage may be optimized for the plant species in question
to improve expression (see Horvath et al referred to above).
[0110] The selectable marker gene and any other parts of the expression construct may be
chosen from those available in the art.
[0111] The nucleic acid construct is incorporated into the plant genome according to conventional
techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic transformation, and
electroporation (
Gasser et al., 1990, Science 244: 1293;
Potrykus, 1990, Bio/Technology 8: 535;
Shimamoto et al., 1989, Nature 338: 274).
[0112] Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice
for generating transgenic dicots (for a review, see
Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), and it can also be used for transforming monocots, although other transformation
methods are generally preferred for these plants. Presently, the method of choice
for generating transgenic monocots, supplementing the Agrobacterium approach, is particle
bombardment (microscopic gold or tungsten particles coated with the transforming DNA)
of embryonic calli or developing embryos (
Christou, 1992, Plant Journal 2: 275-281;
Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162;
Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation
as described by
Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.
[0113] Following transformation, the transformants having incorporated therein the expression
construct are selected and regenerated into whole plants according to methods well-known
in the art.
[0114] The present invention also relates to methods for producing a polypeptide of the
present invention comprising (a) cultivating a transgenic plant or a plant cell comprising
a nucleic acid sequence encoding a protease variant of the present invention under
conditions conducive for production of the protease variant; and (b) recovering the
protease variant.
Animals as Expression Hosts
[0115] The present invention also relates to a transgenic, non-human animal and products
or elements thereof, examples of which are body fluids such as milk and blood, organs,
flesh, and animal cells. Techniques for expressing proteins, e.g. in mammalian cells,
are known in the art, see e.g. the handbook Protein Expression:
A Practical Approach, Higgins and Hames (eds), Oxford University Press (1999), and the three other handbooks in this series relating to Gene Transcription, RNA
processing, and Post-translational Processing. Generally speaking, to prepare a transgenic
animal, selected cells of a selected animal are transformed with a nucleic acid sequence
encoding a protease variant of the present invention so as to express and produce
the protease variant. The protease variant may be recovered from the animal, e.g.
from the milk of female animals, or it may be expressed to the benefit of the animal
itself, e.g. to assist the animal's digestion. Examples of animals are mentioned below
in the section headed Animal Feed and Animal Feed Additives.
[0116] To produce a transgenic animal with a view to recovering the protease variant from
the milk of the animal, a gene encoding the protease variant may be inserted into
the fertilized eggs of an animal in question, e.g. by use of a transgene expression
vector which comprises a suitable milk protein promoter, and the gene encoding the
protease variant. The transgene expression vector is microinjected into fertilized
eggs, and preferably permanently integrated into the chromosome. Once the egg begins
to grow and divide, the potential embryo is implanted into a surrogate mother, and
animals carrying the transgene are identified. The resulting animal can then be multiplied
by conventional breeding. The protease variant may be purified from the animal's milk,
see e.g.
Meade, H.M. et al (1999): Expression of recombinant proteins in the milk of transgenic
animals, Gene expression systems: Using nature for the art of expression. J. M. Fernandez
and J. P. Hoeffler (eds.), Academic Press.
[0117] In the alternative, in order to produce a transgenic non-human animal that carries
in the genome of its somatic and/or germ cells a nucleic acid sequence including a
heterologous transgene construct including a transgene encoding the protease variant,
the transgene may be operably linked to a first regulatory sequence for salivary gland
specific expression of the protease variant, as disclosed in
WO 2000064247.
Animal Feed and Animal Feed Additives
[0118] For the present purposes, the term animal includes all animals, including human beings.
In a particular embodiment, the protease variants and compositions of the invention
can be used as a feed additive for non-human animals. Examples of animals are non-ruminants,
and ruminants, such as sheep, goats, horses, and cattle, e.g. beef cattle, cows, and
young calves. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant
animals include mono-gastric animals, e.g. pigs or swine (including, but not limited
to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including
but not limited to broiler chicks, layers); young calves; and fish (including but
not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including
but not limited to shrimps and prawns).
[0119] The term feed or feed composition means any compound, preparation, mixture, or composition
suitable for, or intended for intake by an animal. The feed can be fed to the animal
before, after, or simultaneously with the diet. The latter is preferred.
[0120] The composition of the invention, when intended for addition to animal feed, may
be designated an animal feed additive. Such additive always comprises the protease
variant in question, preferably in the form of stabilized liquid or dry compositions.
The additive may comprise other components or ingredients of animal feed. The so-called
pre-mixes for animal feed are particular examples of such animal feed additives. Pre-mixes
may contain the enzyme(s) in question, and in addition at least one vitamin and/or
at least one mineral.
[0121] Accordingly, in a particular embodiment, in addition to the component polypeptides,
the composition of the invention may comprise or contain at least one fat-soluble
vitamin, and/or at least one water-soluble vitamin, and/or at least one trace mineral.
Also at least one macro mineral may be included.
[0122] Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin
K, e.g. vitamin K3.
[0123] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1,
vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.
[0124] Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and
cobalt.
[0125] Examples of macro minerals are calcium, phosphorus and sodium.
[0126] Further, optional, feed-additive ingredients are colouring agents, e.g. carotenoids
such as beta-carotene, astaxanthin, and lutein; aroma compounds; stabilizers; polyunsaturated
fatty acids; reactive oxygen generating species; antimicrobial peptides; and/or at
least one additional enzyme.
[0127] Additional enzyme components of the invention include at least one polypeptide having
amylase, preferably alpha-amylase, activity, and/or at least one polypeptide having
xylanase activity; and/or at least one polypeptide having endoglucanase activity;
and/or at least one polypeptide having endo-1,3(4)-beta-glucanase activity; and/or
at least one polypeptide having phytase activity; and/or at least one polypeptide
having galactanase activity; and/or at least one polypeptide having alpha-galactosidase
activity; and/or at least one other polypeptide having protease activity (EC 3.4.-.-
); and/or at least one polypeptide having phospholipase A1 (EC 3.1.1.32), phospholipase
A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), phospholipase C (EC 3.1.4.3), and/or
phospholipase D (EC 3.1.4.4) activity.
[0128] Alpha-amylase activity can be measured as is known in the art, e.g. using a starch-based
substrate.
[0129] Xylanase activity can be measured using any assay, in which a substrate is employed,
that includes 1,4-beta-D-xylosidic endo-linkages in xylans. Different types of substrates
are available for the determination of xylanase activity e.g. Xylazyme cross-linked
arabinoxylan tablets (from MegaZyme), or insoluble powder dispersions and solutions
of azo-dyed arabinoxylan.
[0130] Endoglucanase activity can be determined using any endoglucanase assay known in the
art. For example, various cellulose- or beta-glucan-containing substrates can be applied.
An endoglucanase assay may use AZCL-Barley beta-Glucan, or preferably (1) AZCL-HE-Cellulose,
or (2) Azo-CM-cellulose as a substrate. In both cases, the degradation of the substrate
is followed spectrophotometrically at OD
595 (see the Megazyme method for AZCL-polysaccharides for the assay of endo-hydrolases
at http://www.megazyme.com/booklets/AZCLPOL.pdf.
[0131] Endo-1,3(4)-beta-glucanase activity can be determined using any endo-1,3(4)-beta-glucanase
assay known in the art. A preferred substrate for endo-1,3(4)-beta-glucanase activity
measurements is a cross-linked azo-coloured beta-glucan Barley substrate, wherein
the measurements are based on spectrophotometric determination principles.
[0132] Phytase activity can be measured using any suitable assay, e.g. the FYT assay described
in Example 4 of
WO 98/28408.
[0133] Galactanase can be assayed e.g. with AZCL galactan from Megazyme, and alpha-galactosidase
can be assayed e.g. with pNP-alpha-galactoside.
[0134] For assaying these enzyme activitites the assay-pH and the assay-temperature are
to be adapted to the enzyme in question (preferably a pH close to the optimum pH,
and a temperature close to the optimum temperature). A preferred assay pH is in the
range of 2-10, preferably 3-9, more preferably pH 3 or 4 or 5 or 6 or 7 or 8, for
example pH 3 or pH 7. A preferred assay temperature is in the range of 20-90°C, preferably
30-90°C, more preferably 40-80°C, even more preferably 40-70°C, preferably 40 or 45
or 50°C. The enzyme activity is defined by reference to appropriate blinds, e.g. a
buffer blind.
[0135] Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1,
Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert
Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed
in
WO 03/044049 and
WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
[0136] Examples of antifungal polypeptides (AFP's) are the Aspergillus giganteus, and Aspergillus
niger peptides, as well as variants and fragments thereof which retain antifungal
activity, as disclosed in
WO 94/01459 and
WO 02/090384.
[0137] Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty
acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic
acid.
[0138] Examples of reactive oxygen generating species are chemicals such as perborate, persulphate,
or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
[0139] Usally fat and water soluble vitamins, as well as trace minerals form part of a so-called
premix intended for addition to the feed, whereas macro minerals are usually separately
added to the feed. A premix enriched with a protease of the invention, is an example
of an animal feed additive of the invention.
[0140] In a particular embodiment, the animal feed additive of the invention is intended
for being included (or prescribed as having to be included) in animal diets or feed
at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning
g additive per 100 g feed). This is so in particular for premixes.
[0141] The nutritional requirements of these components (exemplified with poultry and piglets/pigs)
are listed in Table A of
WO 01/58275. Nutritional requirement means that these components should be provided in the diet
in the concentrations indicated.
[0142] In the alternative, the animal feed additive of the invention comprises at least
one of the individual components specified in Table A of
WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and
so forth up to all thirteen, or up to all fifteen individual components. More specifically,
this at least one individual component is included in the additive of the invention
in such an amount as to provide an in-feed-concentration within the range indicated
in column four, or column five, or column six of Table A.
[0143] The present invention also relates to animal feed compositions. Animal feed compositions
or diets have a relatively high content of protein. Poultry and pig diets can be characterised
as indicated in Table B of
WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table
B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.
WO 01/58275 corresponds to
US 09/779334 which is hereby incorporated by reference.
[0144] An animal feed composition according to the invention has a crude protein content
of 50-800 g/kg, and furthermore comprises at least one protease variant as claimed
herein.
[0145] Furthermore, or in the alternative (to the crude protein content indicated above),
the animal feed composition of the invention has a content of metabolisable energy
of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available
phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or
a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine
of 0.5-50 g/kg.
[0146] In particular embodiments, the content of metabolisable energy, crude protein, calcium,
phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one
of ranges 2, 3, 4 or 5 in Table B of
WO 01/58275 (R. 2-5).
[0148] Metabolisable energy can be calculated on the basis of the NRC publication Nutrient
requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition,
committee on animal nutrition, board of agriculture, national research council.
National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy
Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension,
7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen.
ISBN 90-71463-12-5.
[0149] The dietary content of calcium, available phosphorus and amino acids in complete
animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997,
gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen,
Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
[0150] In a particular embodiment, the animal feed composition of the invention contains
at least one protein. The protein may be an animal protein, such as meat and bone
meal, and/or fish meal; or, in a particular embodiment, it may be a vegetable protein.
The term vegetable proteins as used herein refers to any compound, composition, preparation
or mixture that includes at least one protein derived from or originating from a vegetable,
including modified proteins and protein-derivatives. In particular embodiments, the
protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60% (w/w).
[0151] Vegetable proteins may be derived from vegetable protein sources, such as legumes
and cereals, for example materials from plants of the families Fabaceae (Leguminosae),
Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal and
rapeseed meal.
[0152] In a particular embodiment, the vegetable protein source is material from one or
more plants of the family Fabaceae, e.g. soybean, lupine, pea, or bean.
[0153] In another particular embodiment, the vegetable protein source is material from one
or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa.
[0154] Other examples of vegetable protein sources are rapeseed, sunflower seed, cotton
seed, and cabbage.
[0155] Soybean is a preferred vegetable protein source.
[0156] Other examples of vegetable protein sources are cereals such as barley, wheat, rye,
oat, maize (corn), rice, triticale, and sorghum.
[0157] In still further particular embodiments, the animal feed composition of the invention
contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley;
and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25%, preferably 0-10%, fish
meal; 0-25% meat and bone meal; and/or 0-20% whey.
[0158] Animal diets can e.g. be manufactured as mash feed (non pelleted) or pelleted feed.
Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins
and minerals are added according to the specifications for the species in question.
Enzymes can be added as solid or liquid enzyme formulations. For example, a solid
enzyme formulation is typically added before or during the mixing step; and a liquid
enzyme preparation is typically added after the pelleting step. The enzyme may also
be incorporated in a feed additive or premix.
[0159] The final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme
protein per kg diet, for example in the range of 0.5-25 mg enzyme protein per kg animal
diet.
[0160] The protease variant should of course be applied in an effective amount, i.e. in
an amount adequate for improving solubilisation and/or improving nutritional value
of feed. It is at present contemplated that the enzyme is administered in one or more
of the following amounts (dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100;
10-100; 0.05-50; or 0.10-10 - all these ranges being in mg protease enzyme protein
per kg feed (ppm).
[0161] For determining mg enzyme protein per kg feed, the protease is purified from the
feed composition, and the specific activity of the purified protease is determined
using a relevant assay (see under protease activity, substrates, and assays). The
protease activity of the feed composition as such is also determined using the same
assay, and on the basis of these two determinations, the dosage in mg enzyme protein
per kg feed is calculated.
[0162] The same principles apply for determining mg enzyme protein in feed additives. Of
course, if a sample is available of the protease used for preparing the feed additive
or the feed, the specific activity is determined from this sample (no need to purify
the protease from the feed composition or the additive).
Detergent Compositions
[0163] The protease variant of the invention may be added to and thus become a component
of a detergent composition.
[0164] The detergent composition of the invention may for example be formulated as a hand
or machine laundry detergent composition including a laundry additive composition
suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general household hard surface
cleaning operations, or be formulated for hand or machine dishwashing operations.
[0165] In a specific aspect, the invention provides a detergent additive comprising the
protease variant of the invention. The detergent additive as well as the detergent
composition may comprise one or more other enzymes such as another protease, such
as alkaline proteases from Bacillus, a lipase, a cutinase, an amylase, a carbohydrase,
a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an
oxidase, e.g., a laccase, and/or a peroxidase.
[0166] In general the properties of the chosen enzyme(s) should be compatible with the selected
detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic
ingredients, etc.), and the enzyme(s) should be present in effective amounts.
[0167] Suitable lipases include those of bacterial or fungal origin. Chemically modified
or protein engineered mutants are included. Examples of useful lipases include lipases
from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described
in
EP 258068 and
EP 305216 or from H. insolens as described in
WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (
EP 218272), P. cepacia (
EP 331376), P. stutzeri (
GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (
WO 95/06720 and
WO 96/27002), P. wisconsinensis (
WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (
Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (
JP 64/744992) or B. pumilus (
WO 91/16422). Other examples are lipase variants such as those described in
WO 92/05249,
WO 94/01541,
EP 407225,
EP 260105,
WO 95/35381,
WO 96/00292,
WO 95/30744,
WO 94/25578,
WO 95/14783,
WO 95/22615,
WO 97/04079 and
WO 97/07202. Preferred commercially available lipase enzymes include LipolaseTM and Lipolase
UltraTM (Novozymes A/S).
[0168] Suitable amylases (alpha- and/or beta-) include those of bacterial or fungal origin.
Chemically modified or protein engineered mutants are included. Amylases include,
for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis,
described in more detail in
GB 1,296,839. Examples of useful amylases are the variants described in
WO 94/02597,
WO 94/18314,
WO 95/26397,
WO 96/23873,
WO 97/43424,
WO 00/60060, and
WO 01/66712, especially the variants with substitutions in one or more of the following positions:
15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,
264, 304, 305, 391 , 408, and 444. Commercially available amylases are Natalase
™, Supramyl
™, Stainzyme
™, Duramyl
™, Termamyl
™, Fungamyl
™ and BAN
™ (Novozymes A/S), Rapidase
™ and Purastar
™ (from Genencor International Inc.).
[0169] Suitable cellulases include those of bacterial or fungal origin. Chemically modified
or protein engineered mutants are included. Suitable cellulases include cellulases
from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,
e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila
and Fusarium oxysporum disclosed in
US 4,435,307,
US 5,648,263,
US 5,691,178,
US 5,776,757 and
WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour
care benefits. Examples of such cellulases are cellulases described in
EP 0 495257,
EP 531372,
WO 96/11262,
WO 96/29397,
WO 98/08940. Other examples are cellulase variants such as those described in
WO 94/07998,
EP 0 531 315,
US 5,457,046,
US 5,686,593,
US 5,763,254,
WO 95/24471,
WO 98/12307 and
WO 99/01544. Commercially available cellulases include CelluzymTM, and CarezymTM (Novozymes A/S),
ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao
Corporation).
[0170] Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin.
Chemically modified or protein engineered mutants are included. Examples of useful
peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants
thereof as those described in
WO 93/24618,
WO 95/10602, and
WO 98/15257. Commercially available peroxidases include GuardzymeTM (Novozymes).
[0171] The detergent enzyme(s) may be included in a detergent composition by adding separate
additives containing one or more enzymes, or by adding a combined additive comprising
all of these enzymes. A detergent additive of the invention, i.e. a separate additive
or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry,
etc. Preferred detergent additive formulations are granulates, in particular non-dusting
granulates, liquids, in particular stabilized liquids, or slurries.
[0172] Non-dusting granulates may be produced, e.g., as disclosed in
US 4,106,991 and
4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar
weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide
units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon
atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty
acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming
coating materials suitable for application by fluid bed techniques are given in
GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such
as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according
to established methods. Protected enzymes may be prepared according to the method
disclosed in
EP 238216.
[0173] The detergent composition of the invention may be in any convenient form, e.g., a
bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be
aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
[0174] The detergent composition comprises one or more surfactants, which may be non-ionic
including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants
are typically present at a level of from 0.1% to 60% by weight.
[0175] When included therein the detergent will usually contain from about 1% to about 40%
of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate,
alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,
alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
[0176] When included therein the detergent will usually contain from about 0.2% to about
40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate,
alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl
derivatives of glucosamine ("glucamides").
[0177] The detergent may contain 0-65 % of a detergent builder or complexing agent such
as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic
acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl-
or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
[0178] The detergent may comprise one or more polymers. Examples are carboxymethylcellulose,
poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers and lauryl methacrylate/acrylic acid copolymers.
[0179] The detergent may contain a bleaching system which may comprise a H2O2 source such
as perborate or percarbonate which may be combined with a peracid-forming bleach activator
such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively,
the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone
type.
[0180] The enzyme(s) of the detergent composition of the invention may be stabilized using
conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol,
a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g.,
an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl
boronic acid, and the composition may be formulated as described in e.g.
WO 92/19709 and
WO 92/19708.
[0181] The detergent may also contain other conventional detergent ingredients such as e.g.
fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion
agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides,
optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
[0182] It is at present contemplated that in the detergent compositions any enzyme, in particular
the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg
of enzyme protein per liter of wash liqour, preferably 0.05-5 mg of enzyme protein
per liter of wash liqour, in particular 0.1-1 mg of enzyme protein per liter of wash
liqour.
[0183] The enzyme of the invention may additionally be incorporated in the detergent formulations
disclosed in
WO 97/07202.
Method for Generating Protease Variants
[0184] The invention also relates to a method for generating a protease variant of an improved
property, the method comprising the following steps:
(a) selecting a parent protease of at least 60% identity to amino acids 1 to 188 of
SEQ ID NO: 2;
(b) establishing a 3D structure of the parent protease by homology modelling using
the Fig. 2 structure as a model; and/or aligning the parent protease according to
the alignment of Fig. 1;
(c) proposing at least one amino acid substitution, e.g. by:
(i) subjecting the 3D structure of (b) to MD simulations at increased temperatures,
and identifying regions in the amino acid sequence of the parent protease of high
mobility (isotropic fluctuations);
(ii) introducing disulfid bridges by way of cysteine substitutions (C-C);
(iii) introducing proline substitutions (P);
(iv) replacing exposed neutral amino acid residues with negatively charged amino acid
residues (E,D);
(v) replacing exposed neutral amino aicd residues with positively charged amino acid
residues (R,K);
(vi) replacing small amino acid residues inside the protein with bulkier amino acid
residues (W);
(vii) comparing by homology alignment and/or homology modelling according to step
(c)(i) at least two related parent proteases and transferring amino acid residue differences
inbetween these protease backbones, preferably from a backbone having the improved
property to a backbone not having this improved property;
(d) preparing a DNA sequence encoding the parent protease but for inclusion of a DNA
codon of the at least one amino acid substitution proposed in steps (c)(ii)-(c)(vii),
or subjecting the parent DNA sequence to random mutagenesis, targetting at least one
of the regions identified in step (c)(i);
(e) expressing the DNA sequence obtained in step (d) in a host cell, and
(h) selecting a host cell expressing a protease variant with an improved property.
[0185] The invention furthermore relates to a method for producing a protease variant obtainable
or obtained by the method of generating protease variants described above, comprising
(a) cultivating the host cell to produce a supernatant comprising the variant; and
(b) recovering the variant.
[0186] The invention also relates to isolated nucleic acid sequences comprising a nucleic
acid sequence which encodes the protease variant obtainable according to this method,
as well as methods for producing it by (a) cultivating the host cell to produce a
supernatant comprising the variant; and (b) recovering the variant; a transgenic plant,
or plant part, capable of expressing it; transgenic, non-human animals, or products,
or elements thereof, being capable of expressing it; animal feeds, as well as animal
feed additives, comprising it; methods for improving the nutritional value of an animal
feed by use thereof; methods for the treatment of proteins, such as vegetable proteins,
by use thereof; as well s the use thereof (i) in animal feed; (ii) in the preparation
of animal feed; (iii) for improving the nutritional value of animal feed; and/or (iv)
for the treatment of proteins; and/or in detergents.
Alternative Embodiment
[0187] In an alternative embodiment, the term "alteration" is used instead of "substitution"
as the general term for amendments in the protease molecule. This alternative embodiment
includes each of the claims formulated as examplified below for claim 1, and also
specifically includes everything what is stated herein, e.g. definitions (other than
the definition of substitution), i.e. the various aspects, particular embodiments
etc.
[0188] A variant of a parent protease, comprising an alteration in at least one position
of at least one region selected from the group of regions consisting of: 6-18; 22-28;
32-39; 42-58; 62-63; 66-76; 78-100; 103-106; 111-114; 118-131; 134-136; 139-141; 144-151;
155-156; 160-176; 179-181; and 184-188; wherein
- (a) the alteration(s) are independently
(i) an insertion of an amino acid immediately downstream of the position,
(ii) a deletion of the amino acid which occupies the position, and/or
(iii) a substitution of the amino acid which occupies the position;
- (b) the variant has protease activity; and
- (c) each position corresponds to a position of SEQ ID NO: 2, preferably amino acids
1 to 188 thereof; and
- (d) the variant has a percentage of identity to SEQ ID NO: 2, preferably to amino
acids 1 to 188 thereof, of at least 60%.
[0189] The term "polypeptide variant", "protein variant", "enzyme variant", "protease variant"
or simply "variant" refers to a polypeptide of the invention comprising one or more
alteration(s), such as substitution(s), insertion(s), deletion(s), and/or truncation(s)
of one or more specific amino acid residue(s) in one or more specific position(s)
in the polypeptide.
[0190] The term "parent polypeptide", "parent protein", "parent enzyme", "standard enzyme",
"parent protease" or simply "parent" refers to the polypeptide on which the variant
was based. This term also refers to the polypeptide with which a variant is compared
and aligned.
[0191] The term "randomized library", "variant library", or simply "library" refers to a
library of variant polypeptides. Diversity in the variant library can be generated
via mutagenesis of the genes encoding the variants at the DNA triplet level, such
that individual codons are variegated e.g. by using primers of partially randomized
sequence in a PCR reaction. Several techniques have been described, by which one can
create a diverse combinatorial library by variegating several nucleotide positions
in a gene and recombining them, for instance where these positions are too far apart
to be covered by a single (spiked or doped) oligonucleotide primer. These techniques
include the use of in vivo recombination of the individually diversified gene segments
as described in
WO 97/07205 on page 3, lines 8 to 29 (Novozymes A/S). They also include the use of DNA shuffling
techniques to create a library of full length genes, wherein several gene segments
are combined, and wherein each segment may be diversified e.g. by spiked mutagenesis
(
Stemmer, Nature 370, pp. 389-391, 1994 and
US 5,811,238;
US 5,605,793; and
US 5,830,721). One can use a gene encoding a protein "backbone" (wildtype parent polypeptide)
as a template polynucleotide, and combine this with one or more single or double-stranded
oligonucleotides as described in
WO 98/41623 and in
WO 98/41622 (Novozymes A/S). The single-stranded oligonucleotides could be partially randomized
during synthesis. The double-stranded oligonucleotides could be PCR products incorporating
diversity in a specific region. In both cases, one can dilute the diversity with corresponding
segments encoding the sequence of the backbone protein in order to limit the average
number of changes that are introduced.
[0192] Methods have also been established for designing the ratios of nucleotide mixtures
(A; C; T; G) to be inserted in specific codon positions during oligo- or polynucleotide
synthesis, so as to introduce a bias in order to approximate a desired frequency distribution
towards a set of one or more desired amino acids that will be encoded by the particular
codons. It may be of interest to produce a variant library, that comprises permutations
of a number of known amino acid modifications in different locations in the primary
sequence of the polypeptide. These could be introduced post-translationally or by
chemical modification sites, or they could be introduced through mutations in the
encoding genes. The modifications by themselves may previously have been proven beneficial
for one reason or another (e.g. decreasing antigenicity, or improving specific activity,
performance, stability, or other characteristics). In such instances, it may be desirable
first to create a library of diverse combinations of known sequences. For example,
if twelwe individual mutations are known, one could combine (at least) twelwe segments
of the parent protein encoding gene, wherein each segment is present in two forms:
one with, and one without the desired mutation. By varying the relative amounts of
those segments, one could design a library (of size 212) for which the average number
of mutations per gene can be predicted. This can be a useful way of combining mutations,
that by themselves give some, but not sufficient effect, without resorting to very
large libraries, as is often the case when using 'spiked mutagenesis'. Another way
to combine these 'known mutations' could be by using family shuffling of oligomeric
DNA encoding the known mutations with fragments of the full length wild type sequence.
[0193] In describing the various variants produced or contemplated according to the invention,
a number of nomenclatures and conventions are used which are described in detail below.
A frame of reference is first defined by aligning the variant polypeptide with a parent
enzyme. A preferred parent enzyme is Protease 10 (amino acids 1 to 188 of SEQ ID NO:
2). Thereby a number of alterations will be defined in relation to the amino acid
sequence of amino acids 1 to 188 of SEQ ID NO: 2.
[0194] A substitution in a variant is indicated as:
Original amino acid - position - substituted amino acid;
[0195] The three or one letter codes are used, including the codes Xaa and X to indicate
any amino acid residue. Accordingly, the notation "T82S" or "Thr82Ser" means, that
the variant comprises a substitution of threonine with serine in the variant amino
acid position corresponding to the amino acid in position 82 in the parent enzyme,
when the two are aligned as indicated above.
[0196] Where the original amino acid residue may be any amino acid residue, a short hand
notation may at times be used indicating only the position, and the substituted amino
acid, for example:
Position - substituted amino acid; or "82S",
[0197] Such a notation is particular relevant in connection with modification(s) in a series
of homologous polypeptides.
[0198] Similarly when the identity of the substituting amino acid residue(s) is immaterial:
Original amino acid - position; or "T82"
[0199] When both the original amino acid(s) and substituted amino acid(s) may be any amino
acid, then only the position is indicated, e.g.: "82".
[0200] When the original amino acid(s) and/or substituted amino acid(s) may comprise more
than one, but not all amino acid(s), then the amino acids are listed separated by
commas:
Original amino acids - position no. - substituted amino acids; or "T10E,D,Y".
[0201] A number of examples of this nomenclature are listed below:
The substitution of threonine for histidine in position 91 is designated as: "His91Thr"
or "H91T"; or the substitution of any amino acid residue acid for histidine in position
91 is designated as: "His91Xaa" or "H91X" or "His91" or "H91".
[0202] For a modification where the original amino acid(s) and/or substituted amino acid(s)
may comprise more than one, but not all amino acid(s), the substitution of glutamic
acid, aspartic acid, or tyrosine for threonine in position 10:
"Thr10Glu,Asp,Tyr" or "T10E,D,Y"; which indicates the specific variants: "T10E", "T10D",
and "T10Y".
[0203] A deletion of glycine in position 26 will be indicated by: "Glsy26*" or "G26*"
[0204] Correspondingly, the deletion of more than one amino acid residue, such as the deletion
of glycine and glutamine in positions 26 and 27 will be designated "Gly26*+Gln27*"
or "G26*+Q27*"
[0205] The insertion of an additional amino acid residue such as e.g. a lysine after G26
is indicated by: "Gly26GlyLys" or "G26GK"; or, when more than one amino acid residue
is inserted, such as e.g. a Lys, and Ala after G26 this will be indicated as: "Gly26GlyLysAla"
or "G26GKA".
[0206] In such cases the inserted amino acid residue(s) are numbered by the addition of
lower case letters to the position number of the amino acid residue preceding the
inserted amino acid residue(s). In the above example the sequences would thus be:
Parent: |
|
Variant: |
26 |
26 |
26a |
26b |
G |
G |
K |
A |
[0207] In cases where an amino acid residue identical to the existing amino acid residue
is inserted, it is clear that degeneracy in the nomenclature arises. If for example
a glycine is inserted after the glycine in the above example this would be indicated
by "G26GG".
[0208] Given that an alanine were present in position 25, the same actual change could just
as well be indicated as "A25AG":
|
Parent: |
|
Variant: |
Numbering I: |
25 |
26 |
25 |
26 |
26a |
Sequence: |
A |
G |
A |
G |
G |
Numbering II: |
|
|
25 |
25a |
26 |
[0209] Such instances will be apparent to the skilled person, and the indication "G26GG"
and corresponding indications for this type of insertions is thus meant to comprise
such equivalent degenerate indications.
[0210] By analogy, if amino acid sequence segments are repeated in the parent polypeptide
and/or in the variant, it will be apparent to the skilled person that equivalent degenerate
indications are comprised, also when other alterations than insertions are listed
such as deletions and/or substitutions. For instance, the deletion of two consecutive
amino acids "AG" in the sequence "AGAG" from position 194-197, may be written as "A194*+G1956*"
or "A196*+G197*":
|
Parent: |
|
|
|
|
Variant: |
Numbering I: |
194 |
195 |
196 |
197 |
194 |
195 |
Sequence: |
A |
G |
A |
G |
A |
G |
Numbering II: |
|
|
|
|
196 |
197 |
[0211] Variants comprising multiple modifications are separated by pluses, e.g.: "Arg170Tyr+Gly195Glu"
or "R170Y+G195E", representing modifications in positions 170 and 195 substituting
tyrosine and glutamic acid for arginine and glycine, respectively. Thus, "Tyr167Gly,Ala,Ser,Thr+Arg170Gly,Ala,Ser,Thr"
designates the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Gly+Arg170Ser",
"Tyr167Gly+Arg170Thr", "Tyr167 Ala+Arg170Gly", "Tyr167Ala+Arg170Ala", "Tyr167Ala+Arg170Ser",
"Tyr167Ala+Arg170Thr", "Tyr167Ser+Arg170Gly", "Tyr167Ser+Arg170Ala", "Tyr167Ser+Arg170Ser",
"Tyr167Ser+Arg170Thr", "Tyr167Thr+Arg170Gly", "Tyr167Thr+Arg170Ala", "Tyr167Thr+Arg170Ser",
and "Tyr167Thr+Arg170Thr".
[0212] This nomenclature is particular relevant relating to modifications aimed at substituting,
inserting or deleting amino acid residues having specific common properties, such
modifications are referred to as conservative amino acid modification(s).
Various embodiments
[0213] These are additional various embodiments of the invention:
The variant of any one of claims 1-16 and 18-20 which comprises at least one of the
following substitutions: T10Y, A24S, V51T, E53Q, T82S, A86Q, T87S, I96A, G118N, S122R,
N130S, L186I.
[0214] The variant of any one of claims 1-16 and 18-19 which comprises at least one of the
following substitutions: R38T; Q42G,P; R49T,Q; Q54N,R; A89S,T; H91S,T; N92S; S99A,Q;
A120T; E125Q; T129Y,Q; M131L; T135N; Y147F; N151S; R165S; T166V,F; F171Y; V179I,L;
preferably at least one of the following substitutions: R38T; N92S; A120T; E125Q;
M131 L; T135N; Y147F; N151S; R165S; and/or F171Y.
[0215] The variant of any one of claims 1-19 which comprises at least one of the following
substitutions: A25S, T44S, A62S, P95A, V100I, I114V, T176N, N180S, V184L, R185T.
[0216] The variant of any one of claims 1-20 which has amended properties, such as an improved
thermostability and/or a higher or lower optimum temperature, such as a Tm of at least
83.1°C as measured by DSC in 10mM sodium phosphate, 50 mM sodium chloride, pH 7.0.
[0217] The variant of any one of claims 1-20 which derives from a strain of the genus Nocardiopsis,
such as Nocardiopsis alba, Nocardiopsis antarctica, Nocardiopsis prasina, Nocardiopsis
composta, Nocardiopsis dassonvillei, Nocardiopsis exhalans, Nocardiopsis halophila,
Nocardiopsis halotolerans, Nocardiopsis kunsanensis, Nocardiopsis listeri, Nocardiopsis
lucentensis, Nocardiopsis metallicus, Nocardiopsis sp., Nocardiopsis synnemataformans,
Nocardiopsis trehalosi, Nocardiopsis tropica, Nocardiopsis umidischolae, or Nocardiopsis
xinjiangensis, preferably Nocardiopsis alba DSM 15647, Nocardiopsis dassonvillei NRRL
18133, Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235, Nocardiopsis prasina
DSM 15648, Nocardiopsis prasina DSM 15649, Nocardiopsis sp. NRRL 18262, most preferably
Nocardiopsis sp. FERM P-18676.
[0218] A composition, such as an animal feed additive, comprising at least one protease
variant of any one of claims 1-20, and
- (a) at least one fat soluble vitamin;
- (b) at least one water soluble vitamin; and/or
- (c) at least one trace mineral,
optionally further comprising at least one enzyme selected from the following group
of enzymes: amylases, galactanases, alpha-galactosidases, xylanases, endoglucanases,
endo-1,3(4)-beta-glucanases, phytases, phospholipases, and other proteases; if desired
also comprising at least one amylase, and/or phospholipase.
[0219] The present invention is further described by the following examples which should
not be construed as limiting the scope of the invention.
Examples
Example 1: Protease assays
pNA assay
[0220] pNA substrate: Suc-AAPF-pNA (Bachem L-1400).
Temperature : Room temperature (25°C)
Assay buffers :100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl
2, 150mM KCl, 0.01% Triton X-100 adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0,
6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with HCl or NaOH.
20ul protease (diluted in 0.01% Triton X-100) is mixed with 100ul assay buffer. The
assay is started by adding 100ul pNA substrate (50mg dissolved in 1.0ml DMSO and further
diluted 45x with 0.01 % Triton X-100). The increase in OD
405 is monitored as a measure of the protease activity.
Protazyme AK assay
[0221] Substrate : Protazyme AK tablet (cross-linked and dyed casein; from Megazyme)
Temperature : controlled (assay temperature).
Assay buffers :100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl
2, 150mM KCI, 0.01% Triton X-100 adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0,
6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 with HCl or NaOH.
[0222] A Protazyme AK tablet is suspended in 2.0ml 0.01% Triton X-100 by gentle stirring.
500ul of this suspension and 500ul assay buffer are mixed in an Eppendorf tube and
placed on ice. 20ul protease sample (diluted in 0.01% Triton X-100) is added. The
assay is initiated by transferring the Eppendorf tube to an Eppendorf thermomixer,
which is set to the assay temperature. The tube is incubated for 15 minutes on the
Eppendorf thermomixer at its highest shaking rate (1400 rpm). The incubation is stopped
by transferring the tube back to the ice bath. Then the tube is centrifuged in an
icecold centrifuge for a few minutes and 200ul supernatant is transferred to a microtiter
plate. OD
650 is read as a measure of protease activity. A buffer blind is included in the assay
(instead of enzyme).
Example 2: Preparation and Testing of Protease Variants
[0223] Four protease variants comprising the amino acid sequence of amino acids 1 to 188
of SEQ ID NO: 2 (Protease 10) with the single substitutions N47D, T127R, N92K, and
Q54R, respectively, were prepared as described below for variant N47D.
[0225] The N47D variant was constructed by use of the following primers, of which primer
R10WT-CL29 (SEQ ID NO: 11) is gene specific, and primer RSWT126 (SEQ ID NO: 12) mutagenic:
R10WT-CL29: 5' CCGATTATGGAGCGGATTGAACATGCG 3' (SEQ ID NO: 11)
RSWT126: 5' GTGACCATCGGCGACGGCAGGGGCGTCTTCG 3' (SEQ ID NO: 12), to amplify by PCR
an approximately 469 bp DNA fragment from the construct described below.
[0226] The Protease 10 DNA construct used for the above amplification was an expression
cassette (SEQ ID NO: 13) for incorporation into the genome of Bacillus subtilis. The
construct contains a fusion of DNA encoding the signal sequence and the gene encoding
the pro- and the mature protein of Protease 10 (SEQ ID NO: 14), a promoter construction,
and also the cat gene conferring resistance towards chloramphenicol. To facilitate
the integration into the genome by homologous recombination, flanking regions of around
3 kb of a Bacillus subtilis endogenous genes were incorporated upstream and downstream
of the Protease 10 encoding sequence.
[0227] The resulting 469 bp fragment was purified from an agarose gel (Sigma Aldrich cat.no.
A6877) and used as a Mega-primer together with primer R10WT-CL39N (SEQ ID NO: 15)
in a second PCR carried out on the same template.
[0228] R10WT-CL39N: 5' GGAGCTCTGAAAAAAAGGAGAGGATAAAGAATGAA 3' (SEQ ID NO: 15).
[0229] The full construction of approximately 10kb is made in vitro by long range PCR, using
the oligonucleotides R10WT-CL28N (SEQ ID NO: 16), R10WT-CL28C (SEQ ID NO: 17), and
the Expand Long Template PCR System from Roche Applied Science (cat no. 11759060),
according to the suppliers manual.
R10WT-CL28N: 5' GCGTTCCGATAATCGCGGTGACAATGCCG 3' (SEQ ID NO: 16)
R10WT-CL28C: 5' TTCATGAGTCTGCGCCCTGAGATCCTCTG 3' (SEQ ID NO: 17)
[0230] The resulting approximately 1.2 kb fragment was purified and combined in a new PCR
reaction using Expand Long Template PCR System with the flanking fragments of the
construction made by two PCR reactions using R10WT-2C-rev (SEQ ID NO: 18) and R10WT-CL28C
(SEQ ID NO: 17); and RSWT001 (SEQ ID NO: 19) and R10WT-CL28N (SEQ ID NO: 16) as primer
sets. The resulting 10kb fragment can be amplified using the R10WT-CL28N (SEQ ID NO:
16) and R10WT-CL28C (SEQ ID NO: 17) primers, to increase the number of transformants.
R10WT-2C-rev: 5' TAATCGCATGTTCAATCCGCTCCATAATCG 3' (SEQ ID NO: 18)
RSWT001: 5' CCCAACGGTTTCTTCATTCTTTATCCTCTCCTTTTTTTCAGAGC 3' (SEQ ID NO: 19)
[0231] Competent cells of an amylase- and protease-low strain of Bacillus subtilis (such
as strain SHA273 described in
WO92/11357 and
WO95/10603) were transformed with the respective resulting PCR fragments, and chlorampenicol
resistant transformants were selected and checked by DNA sequencing to verify the
presence of the correct mutation on the genome.
[0232] Cells of Bacillus subtilis harbouring constructs encoding Protease 10 and each of
the four variants thereof were used to incubate shakeflasks containing a rich media
(PS-1: 100 g/L Sucrose (Danisco cat.no. 109-0429), 40 g/L crust soy, 10g/L Na
2HPO
4.12H
2O (Merck cat.no. 6579), 0.1ml/L Pluronic PE 6100 (BASF 102-3098)), and cultivation
took place for five days at 30°C under vigorous shaking.
[0233] After cultivation, the supernatants were diluted four times in a 0.2M Na
2HPO
4 buffer, titrated with a 0.1M citric acid to either pH 4.0 or pH 6.0, and split in
two. One half was incubated for four hours at 65°C at the respective pH, after which
it was frozen. The other half was frozen immediately and served as the control.
[0234] Prior to measuring the residual protease activity, the samples were diluted ten times
in 50mM CHES-HEPES buffer, pH 8.5. The activity was determined using a modified version
of the Protazyme AK assay of Example 1, solubilising one tablet of the substrate in
4 ml CHES-HEPES buffer, pH 8.5, mixing under continuous agitation one ml of this substrate
solution with 20ul of diluted protease sample, which was then incubated at 37°C. The
substrate should have the correct temperature prior to adding protease. After 15 minutes
the reaction was stopped by adding 100ul 1M NaOH and the insoluble substrate was precipitated
by centrifugation at 15000 rpm for 3 minutes after which the absorbance at 650nm was
measured. The values should be below OD 3.0, alternatively the protease sample should
be diluted more than ten times prior to the activity measurement.
[0235] The relative residual activity (%) is calculated by dividing the activity after incubation
at 65°C with the activity of the corresponding control. The results of Table 1 below
show that all four variants are of an improved thermostability as compared to Protease
10.
Table 1 Residual activity after incubation for four hours at 65°C
Protease |
% Residual Actitivty pH 6 |
% Residual Activity pH 4 |
Protease 10 + N47D |
44 |
68 |
Protease 10 + T127R |
- |
77 |
Protease 10 + N92K |
- |
55 |
Protease 10 + Q54R |
52 |
67 |
|
|
|
Protease 10 |
19 |
41 |
Example 3: Protease variant 22
[0236] A protease variant designated "Protease 22" and comprising a number of substitutions
in thirteen of the seventeen regions specified in claim 1 was designed. This variant
comprises the following substitutions as compared to the mature part of Protease 10
(amino acids 1-188 of SEQ ID NO: 2): T10Y, A25S, R38T, Q42P, T44S, R49K, Q54R, V56I,
A62S, T82S, S99A, G118Ns, S120T, S122R, E125Q, T129Y, N130S, M131L, R165S, T166A,
F171Y, T176N, V179L, N180S, V184L, and R185T.
[0237] The mature part of Protease 22 is amino acids 1-196 of SEQ ID NO: 21. The DNA sequence
corresponding to SEQ ID NO: 21 is SEQ ID NO: 20.
[0238] The DNA sequence of SEQ ID NO: 20 was constructed and introduced into a Bacillus
host for expression. The expressed protease was purified and characterized as an alpha-lytic
protease (peptidase family S1 E and/or S2A).
[0239] The temperature-activity relationship of Protease 22 was measured at pH9, using the
Protazyme AK assay of Example 1, Protease 10 being included for comparative purposes.
The results are shown in Table 2 below.
Table 2 Temperature profile at pH9 of Protease 22 and Protease 10
|
Relative activity at pH 9 |
|
Temperature (°C) |
Protease 22 |
Protease 10 |
15 |
0.016 |
0.015 |
25 |
0.010 |
0.024 |
37 |
0.028 |
0.068 |
50 |
0.069 |
0.199 |
60 |
0.138 |
0.510 |
70 |
0.474 |
1.000 |
80 |
1.000 |
0,394 |
90 |
0,375 |
- |
[0240] From these results it appears that Protease 22 has a higher temperature optimum at
pH 9 than the Protease 10, viz. around 80°C as compared to around 70°C.
[0241] Differential Scanning Calorimetry (DSC) was used to determine temperature stability
at pH 7.0 of Protease 22 and Protease 10. The purified proteases were dialysed over
night at 4°C against 10 mM sodium phosphate, 50 mM sodium chloride, pH 7.0 and run
on a VP-DSC instrument (Micro Cal) with a constant scan rate of 1.5°C/min from 20
to 100°C. Data-handling was performed using the MicroCal Origin software.
[0242] The resulting denaturation or melting temperatures, Tm's, were: For Protease 22:
83.5°C; for Protease 10: 76.5°C.
[0243] The invention described and claimed herein is not to be limited in scope by the specific
embodiments herein disclosed, since these embodiments are intended as illustrations
of several aspects of the invention. Any equivalent embodiments are intended to be
within the scope of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become apparent to those skilled
in the art from the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. In the case of conflict, the present
disclosure including definitions will control.
1. A variant of a parent protease, comprising a substitution in at least one of the following
positions: 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 22; 23; 24; 25; 26; 27;
28; 32; 33; 34; 35; 36; 37; 38; 39; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53;
54; 55; 56; 57; 58; 62; 63; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 78; 79; 80;
81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 103;
104; 105; 106; 111; 112; 113; 114; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127;
128; 129; 130; 131; 134; 135; 136; 139; 140; 141; 144; 145; 146; 147; 148; 149; 150;
151; 155; 156; 160; 161; 162; 163; 164; 165; 166; 167; 168; 169; 170; 171; 172; 173;
174; 175; 176; 179; 180; 181; 184; 185; 186; 187; and/or 188; wherein
(a) the variant has protease activity; and
(b) each position corresponds to a position of amino acids 1 to 188 of SEQ ID NO:
2; and
(c) the variant has a percentage of identity to amino acids 1 to 188 of SEQ ID NO:
2 of at least 60%.
2. The variant of claim 1 which comprises a substitution in at least one of the following
positions: 6; 7; 8; 9; 10; 12; 13; 16; 17; 18; 22; 23; 24; 25; 26; 27; 28; 32; 33;
37; 38; 39; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 58; 62; 63;
66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87;
88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 103; 105; 106; 111; 113; 114;
118; 120; 122; 124; 125; 127; 129; 130; 131; 134; 135; 136; 139; 140; 141; 144; 145;
146; 147; 148; 149; 150; 151; 155; 156; 160; 161; 162; 163; 164; 165; 166; 167; 168;
169; 170; 171; 172; 173; 174; 175; 176; 179; 180; 181; 184; 185; 186; 187; and/or
188.
3. The variant of claim 2, which comprises at least one of the following substitutions:
6C; 7P; 8C; 9C; 10E,D; 12E,D; 13E,D,P; 16C; 17C; 18C; 22A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y;
23A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; 24C,D,E,F,G,H,I,K,L,M,N,P,Q,R,T,V,W,Y; 25C,D,E,F,G,H,I,K,L,M,N,P,Q,R,T,V,W,Y;
26A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 27A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y;
28A,C,D,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y; 32C; 33C; 37C; 39R,K; 42E,D; 43A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y;
44A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,V,W,Y; 45A,C,D,E,F,G,H,K,L,M,N,P,Q,R,S,T,V,W,Y; 46A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
47A,C,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; 48A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 49A,C,D,E,F,G,H,I,K,L,M,N,P,S,V,W,Y;
50A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 52C; 55C; 56R,K; 58E,D; 62C; 63C; 66A,C,D,E,F,G,H,I,K,L,M,N,P,Q,S,T,V,W,Y;
67A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 68A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y;
69A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,T,V,W,Y; 70A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y;
71A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 72A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y;
73A,C,D,E,F,G,H,I,K,M,N,P,Q,R,S,T,V,W,Y; 74A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y;
75A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; 76C; 78A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,T,V,W,Y;
79A,C,D,E,F,G,H,I,K,L,M,N,P,Q,S,T,V,W,Y; 80A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W;
81A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; 82A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,V,W,Y; 83A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
84A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 85A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W;
86C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y; 87A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,V,W,Y; 88A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,W,Y;
89C,D,E,F,G,H,I,K,L,M,N,P,Q,R,V,W,Y; 90A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 92P,R,
93P; 94C,P; 95E,D; 96E,D,P; 97R,K; 98P; 99R,K; 103C; 105C,P; 106C; 111R,K; 113E,D;
118R,K; 120E,D; 122K; 124R,K; 125P; 127K; 129E,D; 130E,D; 134C; 135P; 136P; 139C;
140E,D; 141C; 144C; 145C; 146C; 147W; 148C; 149C; 150E,D; 151P,E,D; 155C; 156C; 160A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
161A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,T,V,W,Y; 162A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
163A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; 164A,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
165A,C,D,E,F,G,H,I,K,L,M,N,P,Q,T,V,W,Y; 166A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,W,Y; 167A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;
168A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; 169A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y;
170A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y; 172C; 173C; 174P; 175P; 176P; 180R,K; 181R,K;
184P; 187P; and/or 188R,K.
4. The variant of any one of claims 1-3 which comprises at least one of the following
pairs of substitutions: 6C+103C; 8C+105C; 76C+85C; 94C+149C; 55C+63C; 16C+145C; 33C+144C;
62C+173C; 106C+141C; 9C+17C; 18C+156C; 32C+144C; 37C+52C; 67C+71C; 134C+170C; 139C+163C;
146C+148C; and/or 155C+172C.
5. The variant of any one of claims 1-4 which comprises at least one of the following
substitutions: 81P; 82P; 151P; 176P; 24P; 25P; 92P; 93P; 94P; 96P; 98P; 105P; 136P;
184P; 187P; 174P; 7P; 13P; 23P; 27P; 125P; 135P; and/or 175P.
6. The variant of any one of claims 1-4 which comprises at least one of the following
substitutions: 95E,D; 42E,D; 84E,D; 96E,D; 47E; 46E,D; 150E,D; 70E,D; 13E,D; 140E,D;
10E,D; 151E,D; 129E,D; 130E,D; 166E,D; 161E,D; 120E,D; 82E,D; 58E,D; 12E,D; 81E,D;
69E,D; 113E,D; 89E,D; and/or 160E,D.
7. The variant of any one of claims 1-4 which comprises at least one of the following
substitutions: 124R,K; 72R,K; 97R,K; 127K; 56R,K; 122R,K; 181R,K; 180R,K; 25R,K; 92R;
39R,K; 99R,K; 111R,K; 24R,K; 118R,K; 162R,K; and/or 188R,K.
8. The variant of any one of claims 1-4 which comprises at least one of the following
substitutions: 147W; 43W.
9. The variant of any one of claims 1-4 which comprises a substitution in at least one
position of at least one region selected from the group of regions consisting of:
(i) 160-170, 78- 90, 43-50, 66-75, and 22-28;
(ii) 160-170, 78-90, 43-50, and 66-75;
(iii) 160-170, 78-90, and 43-50;
(iv) 160-170, and 78-90; and/or
(v) 160-170.
10. The variant of any one of claims 1-4 which comprises at least one of the following
substitutions: 6C; 8C; 13E,D; 16C; 24P; 25K,P,R; 33C; 42E,D; 46D,E; 47E; 55C; 56R,K;
62C; 63C; 70D,E; 72K,R; 76C; 81P; 82P; 84D,E; 85C; 92P,R, 93P; 94C,P; 95E,D; 96E,D,P;
97R,K; 98P; 103C; 105C,P; 106C; 122R,K; 124R,K; 127K; 136P; 140E,D; 141C; 144C; 145C;
149C; 150E,D; 151P; 173C; 176P; 180R,K; 181R,K; 184P; and/or 187P; preferably 49K,
and/or 166A.
11. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes
the protease variant of any one of claims 1-10.
12. A nucleic acid construct comprising the nucleic acid sequence of claim 11 operably
linked to one or more control sequences that direct the production of the protease
variant in a suitable expression host.
13. A recombinant expression vector comprising the nucleic acid construct of claim 12.
14. A recombinant host cell comprising the nucleic acid construct of claim 12 and/or the
expression vector of claim 13.
15. A method for producing the protease variant of any one of claims 1-10, the method
comprising:
(a) cultivating the host cell of claim 14 to produce a supernatant comprising the
variant; and
(b) recovering the variant.
16. An animal feed additive comprising at least one protease variant of any one of claims
1-10, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.
17. An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising
the protease variant of any one of claims 1-10.
18. A method for improving the nutritional value of an animal feed, wherein the protease
variant of any one of claims 1-10, and/or the composition of any one of claims 16
or 17 is added to the feed.
19. A method for the treatment of proteins, comprising the step of adding the protease
variant of any one of claims 1-10, and/or the composition of any one of claims 16-17
to at least one protein or protein source.
20. Use of the protease variant of any one of claims 1-10, and/or the composition of any
one of claims 16-17 (i) in animal feed; (ii) in the preparation of animal feed; (iii)
for improving the nutritional value of animal feed; and/or (iv) for the treatment
of proteins.
21. Use of the protease variant of any one of claims 1-10 in detergents.